Stabilized Formulations Containing Anti-Ang2 Antibodies

ABSTRACT

The present invention provides pharmaceutical formulations comprising an antibody that specifically binds to angiopoietin 2 (Ang-2). The formulations may contain, in addition to an anti-Ang-2 antibody, at least one amino acid, at least one sugar, or at least one non-ionic surfactant. The pharmaceutical formulations of the present invention exhibit a substantial degree of antibody stability after storage for several months and after being subjected to thermal and other physical stress.

PARENT CASE TEXT

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/589,427 filed on Jan. 23, 2012, the contents of which areincorporated herein by reference in their entirety.

SEQUENCE LISTING

An ST.25 compliant computer readable text file of a sequence listing isfiled concurrently with the present specification according to PCT Rule5.2 and Administrative Instructions Section 802. The contents of thetext file are herein incorporated by reference. A paper copy of thesequence listing, which is identical in content to the ST.25 compliantcomputer readable text file, is included as part of the presentspecification and is herein incorporated by reference.

FIELD

The present invention relates to the field of therapeutic antibodyformulations. More specifically, the present invention relates to thefield of pharmaceutical formulations comprising an antibody thatspecifically binds to angiopoietin-2 (Ang-2).

BACKGROUND

Angiogenesis is the biological process whereby new blood vessels areformed. Aberrant angiogenesis is associated with several diseaseconditions including, e.g., proliferative retinopathies, rheumatoidarthritis, and psoriasis. In addition, it is well established thatangiogenesis is critical for tumor growth and maintenance.Angiopoietin-2 (Ang-2) is a ligand for the Tie-2 receptor (Tie-2) andhas been shown to play a role in angiogenesis. Ang-2 is also referred toin the art as Tie-2 ligand. (U.S. Pat. No. 5,643,755; Yancopoulos etal., 2000, Nature 407:242-248).

Antibodies and other peptide inhibitors that bind to Ang-2 are describedto some extent in, e.g., U.S. Pat. Nos. 6,166,185; 7,521,053; 7,205,275;2006/0018909 and 2006/0246071. There is a need in the art for novelAng-2 modulating agents, including Ang-2 antibodies, that can be used totreat diseases and conditions caused by or exacerbated by angiogenesis.

Therapeutic antibodies must be formulated in a manner that not onlymakes the antibodies suitable for administration to patients, but alsoin a manner that maintains their stability during storage and subsequentuse. For example, therapeutic antibodies in liquid solution are prone todegradation, aggregation, or undesired chemical modifications unless thesolution is formulated properly. The stability of an antibody in liquidformulation depends not only on the kinds of excipients used in theformulation, but also on the amounts and proportions of the excipientsrelative to one another. Furthermore, other considerations aside fromstability must be taken into account when preparing a liquid antibodyformulation. Examples of such additional considerations include theviscosity of the solution and the concentration of antibody that can beaccommodated by a given formulation, and the visual quality or appeal ofthe formulation. Thus, when formulating a therapeutic antibody, greatcare must be taken to arrive at a formulation that remains stable,contains an adequate concentration of antibody, and possesses a suitableviscosity as well as other properties which enable the formulation to beconveniently administered to patients.

Antibodies to the angiopoietin-2 protein (Ang-2) are one example of atherapeutically relevant macromolecule that requires proper formulation.Although some anti-Ang-2 antibodies are known, there nonetheless remainsa need in the art for novel pharmaceutical formulations comprisinganti-Ang-2 antibodies that are sufficiently stable and suitable foradministration to patients.

SUMMARY

The present invention satisfies the aforementioned need by providingpharmaceutical formulations comprising a human antibody thatspecifically binds to human angiopoietin-2 (Ang-2).

In one aspect, a liquid pharmaceutical formulation is provided,comprising: (i) an antibody that specifically binds to angiopoietin-2(Ang-2); (ii) a buffer; (iii) an organic cosolvent; and (iv) astabilizer.

In one embodiment, the antibody is provided at a concentration fromabout 5±0.75 mg/mL to about 150±22.5 mg/mL. In another embodiment, theantibody is provided at a concentration of about 5 mg/ml±0.75 mg/mL. Inanother embodiment, the antibody is provided at a concentration of about25 mg/mL±3.75 mg/mL. In another embodiment, the antibody is provided ata concentration of about 50 mg/mL±7.5 mg/mL.

In some embodiments, exemplary anti-Ang-2 antibodies and Ang-2antigen-binding fragments of the invention comprise HCDR1, HCDR2, HCDR3,LCDR1, LCDR2 and LCDR3 domains, respectively, selected from the groupconsisting of: (i) SEQ ID NO: 4, 6, 8, 12, 14 and 16 (e.g., H1H685);(ii) SEQ ID NO: 28, 30, 32, 36, 38 and 40 (e.g., H1H690); (iii) SEQ IDNO: 52, 54, 56, 60, 62 and 64 (e.g., H1H691); (iv) SEQ ID NO: 148, 150,152, 156, 158 and 160 (e.g., H1H696); (v) SEQ ID NO: 196, 198, 200, 204,206 and 208 (e.g., H1H706); (vi) SEQ ID NO: 268, 270, 272, 276, 278 and280 (e.g., H1M724); and (vii) SEQ ID NO: 436, 438, 440, 444, 446 and 448(e.g., H2M744).

In related embodiments, the invention comprises an anti-Ang-2 antibodyor an antigen-binding fragment of an antibody which specifically bindsAng-2, wherein the antibody or antigen-binding fragment comprises theheavy and light chain CDR domains (i.e., CDR1, CDR2 and CDR3) containedwithin heavy and light chain variable domain sequences selected from thegroup consisting of SEQ ID NO: 2/10, 18/20, 22/24, 26/34, 42/44, 46/48,50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 98/106, 114/116, 118/120,122/130, 138/140, 142/144, 146/154, 162/164, 166/168, 170/178, 186/188,190/192, 194/202, 210/212, 214/216, 218/226, 234/236, 238/240, 242/250,258/260, 262/264, 266/274, 282/284, 286/288, 290/298, 306/308, 310/312,314/322, 330/332, 334/336, 338/346, 354/356, 358/360, 362/370, 378/380,382/384, 386/394, 402/404, 406/408, 410/418, 426/428, 430/432, 434/442,450/452, 454/456, 458/466, 474/476, 478/480, 482/490, 498/500, and502/504. In one embodiment, the antibody or fragment thereof comprisesthe CDR sequences contained within HCVR and LCVR selected from the aminoacid sequence pairs of SEQ ID NO: 18/20, 42/44, 66/68, 162/164, 210/212,266/274, and 434/442.

In one embodiment, the pH of the liquid formulation is about pH 6.0±0.5,pH 6.0±0.4, pH 6.0±0.3, pH 6.0±0.2, pH 6.0±0.1, pH 6.0±0.05, pH6.0±0.01, or pH 6.0. In a specific embodiment, the pH of the liquidformulation is about pH 6.0±0.3. In one embodiment, the liquidpharmaceutical buffer comprises one or more buffers, which has or havean effective buffering range of about pH 5.5 to about pH 7.4, or a pKaof about 6.0.

In one embodiment, the buffer is histidine. In one embodiment, thehistidine is at a concentration of 5 mM±0.75 mM to 50 mM±7.5 mM. In oneembodiment, the histidine is at a concentration of 5 mM±0.75 mM or about5 mM. In one embodiment, the histidine is at a concentration of 10mM±1.5 mM or about 10 mM. In one embodiment, the histidine is at aconcentration of 15 mM±2.25 mM or about 15 mM. In one embodiment, thehistidine is at a concentration of 20 mM±3 mM or about 20 mM. In oneembodiment, the histidine is at a concentration of 25 mM±3.75 mM orabout 25 mM. In one embodiment, the histidine is at a concentration of30 mM±4.5 mM or about 30 mM. In one embodiment, the histidine is at aconcentration of 35 mM±5.25 mM or about 35 mM. In one embodiment, thehistidine is at a concentration of 40 nM±6 mM or about 40 nM. In oneembodiment, the histidine is at a concentration of 45 mM±6.75 mM orabout 45 mM. In one embodiment, the histidine is at a concentration of50 mM±7.5 mM or about 50 mM.

In one embodiment, the organic cosolvent is a nonionic polymercontaining a polyoxyethylene moiety. In some embodiments, the organiccosolvent is any one or more of polysorbate 20, poloxamer 188 andpolyethylene glycol 3350. In a specific embodiment, the organiccosolvent is polysorbate 20.

In one embodiment, the organic cosolvent is at a concentration of fromabout 0.005%±0.00075% to about 1%±0.15% “weight to volume” or “w/v”,wherein, e.g., 0.1 g/ml=10% and 0.01 g/ml=1%. In one embodiment, theorganic cosolvent is polysorbate 20, which is at a concentration ofabout 0.2%±0.03% w/v. In another embodiment, the organic cosolvent ispolysorbate 20, which is at a concentration of 0.01%±0.0015% w/v orabout 0.01% w/v.

In one embodiment, the stabilizer is a sugar. In one embodiment, thesugar is selected from the group consisting of sucrose, mannitol andtrehalose. In a specific embodiment, the stabilizer is sucrose.

In one embodiment, the stabilizer is at a concentration of from 1%±0.15%w/v to 20%±3% w/v. In a specific embodiment, the stabilizer is sucroseat a concentration of 5%±0.75% w/v or about 5% w/v. In another specificembodiment, the stabilizer is sucrose at a concentration of 7.5%±1.125%w/v or about 7.5% w/v. In another specific embodiment, the stabilizer issucrose at a concentration of 10%±1.5% w/v or about 10% w/v. In anotherspecific embodiment, the stabilizer is sucrose at a concentration of12.5%±1.875% w/v or about 12.5% w/v. In another specific embodiment, thestabilizer is sucrose at a concentration of 15%±2.25% w/v or about 15%w/v. In another specific embodiment, the stabilizer is sucrose at aconcentration of 20%±3% w/v or about 20% w/v.

In one embodiment, the viscosity of the formulation is about 1 cPoise toabout 10 cPoise. In one embodiment, the viscosity of the formulation is1.4 cPoise±0.21 cPoise, or about 1.4 cPoise.

In one embodiment, the osmolality of the formulation is within aphysiological range. In one embodiment, the formulation has anosmolality of about 300 milli-Osmoles per kilogram (mOsm) to about 400mOsm. In one embodiment, the osmolality of the formulation is 363mOsm±54 mOsm, or about 363 mOsm.

In one embodiment, at least 96% of the anti-Ang-2 antibody recoveredfrom the liquid pharmaceutical formulation after six months of storageat −80° C. is non-aggregated and un-degraded, as determined by sizeexclusion chromatography. In one embodiment, at least 55% of theanti-Ang-2 antibody recovered from the liquid pharmaceutical formulationafter six months of storage at −80° C. is of the non-basic andnon-acidic form (i.e., main peak or main charge form or “region 2peak”), as determined by ion exchange chromatography.

In one embodiment, at least 96% of the anti-Ang-2 antibody recoveredfrom the liquid pharmaceutical formulation after six months of storageat −30° C. is non-aggregated and un-degraded, as determined by sizeexclusion chromatography. In one embodiment, at least 55% of theanti-Ang-2 antibody recovered from the liquid pharmaceutical formulationafter six months of storage at −30° C. is of the main charge form, asdetermined by ion exchange chromatography.

In one embodiment, at least 96% of the anti-Ang-2 antibody recoveredfrom the liquid pharmaceutical formulation after six months of storageat −20° C. is non-aggregated and un-degraded, as determined by sizeexclusion chromatography. In one embodiment, at least 55% of theanti-Ang-2 antibody recovered from the liquid pharmaceutical formulationafter six months of storage at −20° C. is of the main charge form, asdetermined by ion exchange chromatography.

In one embodiment, at least 96% of the anti-Ang-2 antibody recoveredfrom the liquid pharmaceutical formulation after nine months of storageat 5° C. is of the non-aggregated and un-degraded form, as determined bysize exclusion chromatography. In one embodiment, at least 56% of theanti-Ang-2 antibody recovered from the liquid pharmaceutical formulationafter nine months of storage at 5° C. is of the main charge form, asdetermined by ion exchange chromatography.

In one embodiment, at least 98% of the anti-Ang-2 antibody recoveredfrom the liquid pharmaceutical formulation after three months of storageat 25° C. is of the non-aggregated and un-degraded form, as determinedby size exclusion chromatography. In one embodiment, at least 54% of theanti-Ang-2 antibody recovered from the liquid pharmaceutical formulationafter three months of storage at 25° C. is of the main charge form, asdetermined by ion exchange chromatography.

In one embodiment, at least 97% of the anti-Ang-2 antibody recoveredfrom the liquid pharmaceutical formulation after one month of storage at37° C. is of the non-aggregated and un-degraded form, as determined bysize exclusion chromatography. In one embodiment, at least 47% of theanti-Ang-2 antibody recovered from the liquid pharmaceutical formulationafter one month of storage at 37° C. is of the main charge form, asdetermined by ion exchange chromatography.

In one embodiment, at least 95% of the anti-Ang-2 antibody recoveredfrom the liquid pharmaceutical formulation after 28 days of storage at45° C. is of the non-aggregated and un-degraded form, as determined bysize exclusion chromatography. In one embodiment, at least 32% of theanti-Ang-2 antibody recovered from the liquid pharmaceutical formulationafter 28 days of storage at 45° C. is of the main charge form, asdetermined by ion exchange chromatography.

In one aspect, a liquid pharmaceutical formulation is provided,comprising: (i) from 5±0.75 mg/ml to 150±22.5 mg/ml of a human antibodythat specifically binds to human Ang-2; (ii) from 5 mM±0.75 mM to 50mM±7.5 mM histidine; (iii) from 0.005%±0.000075% to 1%±0.15% (w/v)polysorbate 20; and (iv) from 1%±0.15% to 20%±3% (w/v) sucrose, at a pHof from about 5.5 to about 6.5. The anti-Ang-2 antibody of this aspectcomprises a heavy chain variable region (HCVR) and a light chainvariable region (LCVR) such that the HCVR/LCVR combination comprisesheavy and light chain complementarity determining regions(HCDR1-HCDR2-HCDR3/LCDR1-LCDR2-LCDR3), which comprise the amino acidsequences of SEQ ID NOs:4-6-8/SEQ ID NOs:12-14-16, respectively. In aparticular embodiment, the anti-Ang-2 antibody comprises a heavy chainvariable region (HCVR) and light chain variable region (LCVR) comprisingan amino acid sequence of SEQ ID NO: 18 and SEQ ID NO: 20, respectively(antibody HIH685P of U.S. Pub. Pat. App. No. 20110027286, which isspecifically incorporated herein by reference in its entirety).

In another embodiment, the liquid formulation comprises (i) 50±7.5 mg/mLof HIH685P; (ii) 10±1.5 mM histidine; (iii) 0.2%±0.03% (w/v) polysorbate20; and (iv) 10%±1.5% (w/v) sucrose, at a pH of 6.0±0.5.

In one embodiment of this aspect, the liquid formulation comprises (i)25±3.75 mg/mL of HIH685P; (ii) 10±1.5 mM histidine; (iii) 0.2%±0.03%(w/v) polysorbate 20; and (iv) 10%±1.5% (w/v) sucrose, at a pH of6.0±0.5. In one embodiment of this particular formulation, after storageof the formulation at 45° for 28 days, ≧95% of the antibody is nativeand ≧32% of the antibody is of the main charge form. In one embodimentof this particular formulation, after storage of the formulation at 37°for one month, ≧97% of the antibody is native and ≧47% of the antibodyis of the main charge form. In one embodiment of this particularformulation, after storage of the formulation at 25° for three months,≧98% of the antibody is native and ≧54% of the antibody is of the maincharge form. In one embodiment of this particular formulation, afterstorage of the formulation at 5° for nine months, ≧96% of the antibodyis native and ≧56% of the antibody is of the main charge form. In oneembodiment of this particular formulation, after storage of theformulation at −20° for three months, ≧9% of the antibody is native and≧55% of the antibody is of the main charge form. In one embodiment ofthis particular formulation, after storage of the formulation at −30°for six months, ≧96% of the antibody is native and ≧55% of the antibodyis of the main charge form. In one embodiment of this particularformulation, after storage of the formulation at −80° for six months,≧96% of the antibody is native and ≧55% of the antibody is of the maincharge form.

In one aspect, a liquid pharmaceutical formulation of any of thepreceding aspects is provided in a container. In one embodiment, thecontainer is a polycarbonate vial. In another embodiment, the containeris a glass vial. In one embodiment, the glass vial is a type 1borosilicate glass vial with a fluorocarbon-coated butyl rubber stopper.In another embodiment, the container is a microinfuser. In anotherembodiment, the container is a syringe. In a specific embodiment, thesyringe comprises a fluorocarbon-coated plunger. In one specificembodiment, the syringe is a 1 mL long glass syringe containing lessthan about 500 parts per billion of tungsten equipped with a 27-Gneedle, a fluorocarbon-coated butyl rubber stopper, and a latex-free,non-cytotoxic rubber tip cap. In a more specific embodiment, the syringeis a NUOVA OMPI 1 mL long glass syringe equipped with a 27-G thin wallneedle, a FLUROTEC-coated 4023/50 rubber stopper, and a FM 27 rubber tipcap. In another specific embodiment, the syringe is a 1 mL or 3 mLplastic syringe fitted with a 27-G needle. In a more specificembodiment, the plastic syringe is distributed by BECTON DICKINSON. Inone embodiment, the container is a polyvinyl chloride IV bag. In anotherembodiment, the container is a polyolefin IV bag.

In one aspect, a pharmaceutical formulation comprising (a) 50 mg/mL±7.5mg/mL of an anti-Ang-2 antibody, (b) 10 mM±1.5 mM histidine, pH 6±0.5,(c) 0.2% w/v±0.03% polysorbate 20, and (d) 10% w/v±1.5% sucrose isprovided, wherein (a) the antibody comprises an HCVD of SEQ ID NO: 18and an LCVD of SEQ ID NO: 20, (b) over 96% of the antibodies in theformulation have a molecular weight of about 150.9 kDa±1 kDa, (c) atleast 53% of the antibodies in the formulation have an isoelectric pointof about 8.13±0.01, (d) from about 90% to about 92% of the antibodies inthe formulation are fucosylated, and (e) about 2.5% of the heavy chainsof the antibodies lack a C-terminal lysine.

In one embodiment, the pharmaceutical formulation consists of (a) 50mg/mL±7.5 mg/mL of an anti-Ang-2 antibody, (b) 10 mM±1.5 mM histidine,pH 6±0.5, (c) 0.2% w/v±0.03% polysorbate 20, and (d) 10% w/v±1.5%sucrose is provided, wherein (a) the antibody comprises an HCVD of SEQID NO: 18 and an LCVD of SEQ ID NO: 20, (b) over 96% of the antibodiesin the formulation have a molecular weight of about 150.9 kDa±1 kDa, (c)at least 53% of the antibodies in the formulation have an isoelectricpoint of about 8.13±0.01, (d) from about 90% to about 92% of theantibodies in the formulation are fucosylated, and (e) about 2.5% of theheavy chains of the antibodies lack a C-terminal lysine.

In one aspect, a pharmaceutical formulation comprising (a) 25 mg/mL±3.75mg/mL of an anti-Ang-2 antibody, (b) 10 mM±1.5 mM histidine, pH 6±0.5,(c) 0.2% w/v±0.03% polysorbate 20, and (d) 10% w/v±1.5% sucrose isprovided, wherein (a) the antibody comprises an HCVD of SEQ ID NO: 18and an LCVD of SEQ ID NO: 20, (b) over 96% of the antibodies in theformulation have a molecular weight of about 150.9 kDa±1 kDa, (c) atleast 53% of the antibodies in the formulation have an isoelectric pointof about 8.13±0.01, (d) from about 90% to about 92% of the antibodies inthe formulation are fucosylated, and (e) about 2.5% of the heavy chainsof the antibodies lack a C-terminal lysine.

In one embodiment, the pharmaceutical formulation consists of (a) 25mg/mL±3.75 mg/mL of an anti-Ang-2 antibody, (b) 10 mM±1.5 mM histidine,pH 6±0.5, (c) 0.2% w/v±0.03% polysorbate 20, and (d) 10% w/v±1.5%sucrose is provided, wherein (a) the antibody comprises an HCVD of SEQID NO: 18 and an LCVD of SEQ ID NO: 20, (b) over 96% of the antibodiesin the formulation have a molecular weight of about 150.9 kDa±1 kDa, (c)at least 53% of the antibodies in the formulation have an isoelectricpoint of about 8.13±0.01, (d) from about 90% to about 92% of theantibodies in the formulation are fucosylated, and (e) about 2.5% of theheavy chains of the antibodies lack a C-terminal lysine.

In one aspect, a kit comprising a pharmaceutical composition of any oneof the preceding aspects, a container, and instructions is provided. Inone embodiment, the container is a prefilled syringe. In one embodiment,the container is a borosilicate vial fitted with a FLUROTEC-coated4023/50 rubber stopper.

Other embodiments of the present invention will become apparent from areview of the ensuing detailed description.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about”, when used in reference to a particular recited numerical valueor range of values, means that the value may vary from the recited valueby no more than 2%. For example, as used herein, the expression “about100” includes 98 and 102 and all values in between (e.g., 98.00, 98.01,98.02, 98.03, 98.04, . . . , 101.96, 101.97, 101.98, 101.99, 102.00).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpublications mentioned herein are incorporated herein by reference todescribe in their entirety.

Pharmaceutical Formulations

As used herein, the expression “pharmaceutical formulation” means acombination of at least one active ingredient (e.g., a small molecule,macromolecule, compound, etc. which is capable of exerting a biologicaleffect in a human or non-human animal), and at least one inactiveingredient which, when combined with the active ingredient or one ormore additional inactive ingredients, is suitable for therapeuticadministration to a human or non-human animal. The term “formulation”,as used herein, means “pharmaceutical formulation” unless specificallyindicated otherwise. The present invention provides pharmaceuticalformulations comprising at least one therapeutic polypeptide. Accordingto certain embodiments of the present invention, the therapeuticpolypeptide is an antibody, or an antigen-binding fragment thereof,which binds specifically to human angiopoietin-2 (Ang-2) protein. Morespecifically, the present invention includes pharmaceutical formulationsthat comprise: (i) a human antibody that specifically binds to humanAng-2 (ii) a histidine buffer; (iii) an organic cosolvent that is anon-ionic surfactant; and (iv) a thermal stabilizer that is acarbohydrate. Specific exemplary components and formulations includedwithin the present invention are described in detail below.

Antibodies that Bind Specifically to Ang-2

The pharmaceutical formulations of the present invention may comprise ahuman antibody, or an antigen-binding fragment thereof, that bindsspecifically to human Ang-2. As used herein, the term “Ang-2” or “ANG2”means a human angiopoietin-2, which is generally known as an autocrineantagonist of Tie2 activation. Ang-2 is generally known in the art to“prime” the vascular endothelium to receive the effects of cytokines.Ang-2 is strongly expressed in tumor vasculature, and is generallythought to act synergistically with other cytokines (i.e., vascularendothelial growth factor) to promote angiogenesis and tumorprogression. An exemplary human Ang-2 amino acid sequence is describedin SEQ ID NO: 518. Antibodies to human Ang-2 are described in patentapplication publications US 2010/0166768, US 2011/0065902, WO2010/077854, and US 2011/0027286, which are herein incorporated byreference.

The term “antibody”, as used herein, is generally intended to refer toimmunoglobulin molecules comprising four polypeptide chains: two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds,as well as multimers thereof (e.g., IgM); however, immunoglobulinmolecules consisting of only heavy chains (i.e., lacking light chains)are also encompassed within the definition of the term “antibody”. Eachheavy chain comprises a heavy chain variable region (abbreviated hereinas HCVR or V_(H)) and a heavy chain constant region. The heavy chainconstant region comprises three domains, CH1, CH2 and CH3. Each lightchain comprises a light chain variable region (abbreviated herein asLCVR or V_(L)) and a light chain constant region. The light chainconstant region comprises one domain (CL1). The V_(H) and V_(L) regionscan be further subdivided into regions of hypervariability, termedcomplementary determining regions (CDRs), interspersed with regions thatare more conserved, termed framework regions (FR). Each V_(H) and V_(L)is composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

Unless specifically indicated otherwise, the term “antibody”, as usedherein, shall be understood to encompass complete antibody molecules aswell as antigen-binding fragments thereof. The term “antigen-bindingportion” or “antigen-binding fragment” of an antibody (or simply“antibody portion” or “antibody fragment”), as used herein, refers toone or more fragments of an antibody that retain the ability tospecifically bind to human Ang-2 or an epitope thereof.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds human Ang-2 is substantially free of antibodies that specificallybind antigens other than human Ang-2).

The term “specifically binds”, or the like, means that an antibody orantigen-binding fragment thereof forms a complex with an antigen that isrelatively stable under physiologic conditions. Specific binding can becharacterized by a dissociation constant of at least about 1×10⁻⁶M orgreater. Methods for determining whether two molecules specifically bindare well known in the art and include, for example, equilibriumdialysis, surface plasmon resonance, and the like. An isolated antibodythat specifically binds human Ang-2 may, however, have cross-reactivityto other antigens, such as Ang-2 molecules from other species(orthologs). In the context of the present invention, multispecific(e.g., bispecific) antibodies that bind to human Ang-2 as well as one ormore additional antigens are deemed to “specifically bind” human Ang-2.Moreover, an isolated antibody may be substantially free of othercellular material or chemicals.

Exemplary anti-human Ang-2 antibodies that may be included in thepharmaceutical formulations of the present invention are set forth inpatent application publications US 2010/0166768, US 2011/0065902, US2011/0027286 and WO 2010/077854, the disclosures of which areincorporated herein by reference in their entirety.

According to certain embodiments of the present invention, theanti-human Ang-2 HIH685P antibody is a human IgG1 comprising a heavychain variable region that is of the IGHV3-13.01 subtype and a lightchain variable region that is of the IGKV3-20.01 subtype (see Barbie andLefranc, The Human Immunoglobulin Kappa Variable (IGKV) Genes andJoining (IGKJ) Segments, Exp. Clin. Immunogenet. 1998; 15:171-183; andScaviner, D. et al., Protein Displays of the Human Immunoglobulin Heavy,Kappa and Lambda Variable and Joining Regions, Exp. Clin. Immunogenet.,1999; 16:234-240). The germline IGHV3-13 and IGKV3-20 sequences, and theamino acid position assignment numbers presented herein comport with theinternational Immunogenetics (IMGT) information system, as described inLefranc, M.-P., et al., IMGT®, the international ImMunoGeneTicsinformation System®, Nucl. Acids Res, 37, D1006-D1012 (2009).

In some embodiments, the anti-human Ang-2 HIH685P comprises at least oneamino acid substitution relative to the canonical heavy chain variableregion, which results in a change in the angle of rotation of thepeptide chain within a CDR, which is reasonably expected to alter theexposed surface of the antibody relative to the germline IGHV3-13sequence. In some embodiments, the amino acid substitution comprises thesubstitution of proline for the isoleucine at position 39 within CDR2 ofIGHV3-13.

In some embodiments, the anti-human Ang-2 HIH685P antibody comprises atleast one amino acid substitution, which creates a charge change withinthe third CDR of the germline IGKV3-20. In some embodiments, the aminoacid substitution or substitutions are selected from the groupconsisting of (a) a basic amino acid substituted for an uncharged polaramino acid within CDR3 (e.g., at position 106) of IGHV3-20, and (b) anacidic amino acid substituted for an uncharged polar amino acid withinCDR3 (e.g., at position 108) of IGKV3-20. Changes in the charge displayat the CDR surface is expected to affect the antibody's interface withthe solvent, and thus create unpredictable conditions for maintaining oradvancing the stability of the antibody in solution.

According to certain embodiments of the present invention, theanti-human Ang-2 antibody, or antigen-binding fragment thereof,comprises a heavy chain complementary determining region (HCDR) 1 of SEQID NO: 4, an HCDR2 of SEQ ID NO: 6, and an HCDR3 of SEQ ID NO: 8. Incertain embodiments, the anti-human Ang-2 antibody, or antigen-bindingfragment thereof, comprises an HCVD of SEQ ID NO: 18.

According to certain embodiments of the present invention, theanti-human Ang-2, or antigen-binding fragment thereof, comprises a light(kappa) chain complementary determining region (LCDR) 1 of SEQ ID NO:12, an LCDR2 of SEQ ID NO: 14, and an LCDR3 of SEQ ID NO: 16. In certainembodiments, the anti-human Ang-2 antibody, or antigen-binding fragmentthereof, comprises an LCVD of SEQ ID NO: 20.

The non-limiting, exemplary antibody used in the Examples herein isreferred to as “HIH685P”, as in US 2011/0027286. This antibody comprisesan HCVR/LCVR amino acid sequence pair having SEQ ID NOs: 18/20, andHCDR1-HCDR2-HCDR3/LCDR1-LCDR2-LCDR3 domains represented by SEQ ID NOs:4-6-8/SEQ ID NOs: 12-14-16.

The amount of antibody, or antigen-binding fragment thereof, containedwithin the pharmaceutical formulations of the present invention may varydepending on the specific properties desired of the formulations, aswell as the particular circumstances and purposes for which theformulations are intended to be used. In certain embodiments, thepharmaceutical formulations are liquid formulations that may contain5±0.75 mg/mL to 150±22.5 mg/mL of antibody; 7.5±1.125 mg/mL to 140±21mg/mL of antibody; 10±1.5 mg/mL to 130±19.5 mg/mL of antibody;12.5±1.875 mg/mL to 120±18 mg/mL of antibody; 15±2.25 mg/mL to 110±16.5mg/mL of antibody; 17.5±2.625 mg/mL to 100±15 mg/mL of antibody; 20±3mg/mL to 90±13.5 mg/mL of antibody; 22.5±3.375 mg/mL to 80±12 mg/mL ofantibody; 25±3.75 mg/mL to 70±10.5 mg/mL of antibody; 27.5±4.125 mg/mLto 60±9 mg/mL of antibody; 30±4.5 mg/mL to 50±7.5 mg/mL of antibody;25±3.75 mg/mL of antibody; or 50±7.5 mg/ml. For example, theformulations of the present invention may comprise about 20 mg/mL; about25 mg/mL; about 30 mg/mL; about 35 mg/mL; about 40 mg/mL; about 45mg/mL; about 50 mg/mL; about 55 mg/mL; or about 60 mg/mL of an antibodyor an antigen-binding fragment thereof, that binds specifically to humanAng-2.

Excipients and pH

The pharmaceutical formulations of the present invention comprise one ormore excipients. The term “excipient”, as used herein, means anynon-therapeutic agent added to the formulation to provide a desiredconsistency, viscosity or stabilizing effect.

In certain embodiments, the pharmaceutical formulation of the inventioncomprises at least one organic cosolvent in a type and in an amount thatstabilizes the human Ang-2 antibody under conditions of rough handlingor agitation, such as, e.g., vortexing. In some embodiments, what ismeant by “stabilizes” is the prevention of the formation of more than 4%aggregated antibody of the total amount of antibody (on a molar basis)over the course of rough handling. In some embodiments, rough handlingis vortexing a solution containing the antibody and the organiccosolvent for about 60 minutes or about 120 minutes.

In certain embodiments, the organic cosolvent is a non-ionic surfactant,such as an alkyl poly(ethylene oxide). Specific non-ionic surfactantsthat can be included in the formulations of the present inventioninclude, e.g., polysorbates such as polysorbate 20, polysorbate 28,polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80,polysorbate 81, and polysorbate 85; poloxamers such as poloxamer 181,poloxamer 188, poloxamer 407; or polyethylene glycol (PEG). Polysorbate20 is also known as TWEEN 20, sorbitan monolaurate andpolyoxyethylenesorbitan monolaurate. Poloxamer 188 is also known asPLURONIC F68.

The amount of non-ionic surfactant contained within the pharmaceuticalformulations of the present invention may vary depending on the specificproperties desired of the formulations, as well as the particularcircumstances and purposes for which the formulations are intended to beused. In certain embodiments, the formulations may contain 0.01%±0.0015%to 1%±0.15% surfactant. For example, the formulations of the presentinvention may comprise about 0.0085%; about 0.01%; about 0.02%; about0.03%; about 0.04%; about 0.05%; about 0.06%; about 0.07%; about 0.08%;about 0.09%; about 0.1%; about 0.11%; about 0.12%; about 0.13%; about0.14%; about 0.15%; about 0.16%; about 0.17%; about 0.18%; about 0.19%;about 0.20%; about 0.21%; about 0.22%; about 0.23%; about 0.24%; about0.25%; about 0.3%; about 0.4%; about 0.5%; about 0.6%; about 0.7%; about0.8%; about 0.9%; about 1%; about 1.1%; about 1.15%; or about 1.2%polysorbate 20 or poloxamer 188.

The pharmaceutical formulations of the present invention may alsocomprise one or more stabilizers in a type and in an amount thatstabilizes the human Ang-2 antibody under conditions of thermal stress.In some embodiments, what is meant by “stabilizes” is maintaininggreater than about 93% of the antibody in a native conformation when thesolution containing the antibody and the thermal stabilizer is kept atabout 45° C. for up to about 28 days. In some embodiments, what is meantby “stabilizes” is wherein less than about 4% of the antibody isaggregated when the solution containing the antibody and the thermalstabilizer is kept at about 45° C. for up to about 28 days. In someembodiments, what is meant by “stabilizes” is maintaining greater thanabout 96% of the antibody in a native conformation when the solutioncontaining the antibody and the thermal stabilizer is kept at about 37°C. for up to about 28 days. In some embodiments, what is meant by“stabilizes” is wherein less than about 2% of the antibody is aggregatedwhen the solution containing the antibody and the thermal stabilizer iskept at about 37° C. for up to about 28 days. As used herein, “native”means the major form of the antibody by size exclusion, which isgenerally an intact monomer of the antibody.

In certain embodiments, the thermal stabilizer is a sugar or sugaralcohol selected from sucrose, sorbitol, glycerol, trehalose andmannitol, or any combination thereof, the amount of which containedwithin the formulation can vary depending on the specific circumstancesand intended purposes for which the formulation is used. In certainembodiments, the formulations may contain about 3% to about 20% sugar orsugar alcohol; about 4% to about 19% sugar or sugar alcohol; about 5% toabout 18% sugar or sugar alcohol; about 6% to about 17% sugar or sugaralcohol; about 7% to about 16% sugar or sugar alcohol; about 8% to about15% sugar or sugar alcohol; about 9% to about 16% sugar or sugaralcohol; about 7% to about 13% sugar or sugar alcohol; about 8% to about12% sugar or sugar alcohol; about 9% to about 11% sugar or sugaralcohol; or about 10% sugar or sugar alcohol. For example, thepharmaceutical formulations of the present invention may comprise4%±0.6%; 5%±0.75%; 6%±0.9%; 7%±1.05%; 8%±1.2%; 9%±1.35%; 10%±1.5%;11%±1.65%; 12%±1.8%; 13%±1.95%; or about 14%±2.1% sugar or sugar alcohol(e.g., sucrose, trehalose or mannitol).

The pharmaceutical formulations of the present invention may alsocomprise a buffer or buffer system, which serves to maintain a stable pHand to help stabilize the human Ang-2 antibody. In some embodiments,what is meant by “stabilizes” is wherein less than 5%±0.5% or no morethan about 4.3% of the antibody is aggregated when the solutioncontaining the antibody and the buffer is kept at about 45° C. for up toabout 28 days. In some embodiments, what is meant by “stabilizes” iswherein at least 92%±0.5% of the antibody is in its native conformationas determined by size exclusion chromatography when the solutioncontaining the antibody and the buffer is kept at about 45° C. for up toabout 28 days. By “native” or “native conformation”, what is meant isthe antibody fraction that is not aggregated or degraded. This isgenerally determined by an assay that measures the relative size of theantibody entity, such as a size exclusion chromatographic assay. Thenon-aggregated and non-degraded antibody elutes at a fraction thatequates to the native antibody, and is generally the main elutionfraction. Aggregated antibody elutes at a fraction that indicates a sizegreater than the native antibody. Degraded antibody elutes at a fractionthat indicates a size less than the native antibody.

In some embodiments, what is meant by “stabilizes” is wherein at least52%±0.5% of the antibody is in its main charge form as determined bycation exchange chromatography when the solution containing the antibodyand the buffer is kept at about 45° C. for up to about 28 days. By “maincharge” or “main charge form”, what is meant is the fraction of antibodythat elutes from an ion exchange resin in the main peak, which isgenerally flanked by more “basic” peaks on one side and more “acidic”peaks on the other side.

The pharmaceutical formulations of the present invention may have a pHof from about 5.5 to about 6.5. For example, the formulations of thepresent invention may have a pH of about 5.5; about 5.6; about 5.7;about 5.8; about 5.9; about 6.0; about 6.1; about 6.2; about 6.3; about6.4; or about 6.5. In some embodiments, the pH is 6.0±0.4; 6.0±0.3;6.0±0.2; 6.0±0.1; about 6.0; or 6.0.

In some embodiments, the buffer or buffer system comprises at least onebuffer that has a buffering range that overlaps fully or in part therange of pH 5.5-7.4. In one embodiment, the buffer has a pKa of about6.0±0.5. In certain embodiments, the buffer comprises a histidinebuffer. In certain embodiments, the histidine is present at aconcentration of 5 mM±0.75 mM to 15 mM±2.25 mM; 6 mM±0.9 mM to 14 mM±2.1mM; 7 mM±1.05 mM to 13 mM±1.95 mM; 8 mM±1.2 mM to 12 mM±1.8 mM; 9mM±1.35 mM to 11 mM±1.65 mM; 10 mM±1.5 mM; or about 10 mM. In certainembodiments, the buffer system comprises histidine at 10 mM±1.5 mM, at apH of 6.0±0.3 or 6.4±0.3.

Exemplary Formulations

According to one aspect of the present invention, the pharmaceuticalformulation is a low viscosity, i.e, having a viscosity of under 10cPoise or about 1.4±0.21 cPoise, generally physiologically isotonic,i.e., between 300 and 400 mOsm or about 363±54 mOsm, liquid formulation,which comprises: (i) a human antibody that specifically binds to humanAng-2 (e.g., HIH685P), at a concentration of 25 mg/mL±3.75 mg/mL, or 50mg/mL±7.5 mg/mL; (ii) a buffer system that provides sufficient bufferingat about pH 6.0±0.3; (iii) a sugar which serves as a thermal stabilizer;and (iv) an organic cosolvent, which protects the structural integrityif the antibody.

According to one embodiment, the pharmaceutical formulation comprises:(i) a human IgG1 antibody that specifically binds to human Ang-2 andwhich comprises a substituted IGHV3-13.01 type heavy chain variableregion and a substituted IGKV3-20.01 type light chain variable region(e.g., HIH685P) at a concentration from 20±3 mg/mL to about 60±9 mg/mL;(ii) a buffer system comprising histidine, which buffers effectively atabout pH 6.0±0.3; (iii) sucrose; and (iv) a non-ionic detergent, such asa polysorbate.

According to one embodiment, the pharmaceutical formulation comprises:(i) a human IgG1 antibody that specifically binds to human Ang-2, andwhich comprises an HCDR1 of SEQ ID NO: 4, an HCDR2 of SEQ ID NO: 6, anHCDR3 of SEQ ID NO: 8, an LCDR1 of SEQ ID NO: 14, an LCDR2 of SEQ ID NO:14, and an LCDR3 of SEQ ID NO: 16, at a concentration of 25 mg/ml±3.75mg/mL; (ii) histidine at 10 mM±1.5 mM, which buffers at pH 6.0±0.3;(iii) sucrose at 10% w/v±1.5% w/v; and (iv) polysorbate 20 at 0.2%w/v±0.03% w/v.

According to one embodiment, the pharmaceutical formulation comprises:(i) a human IgG1 antibody that specifically binds to human Ang-2, andwhich comprises an HCDR1 of SEQ ID NO: 4, an HCDR2 of SEQ ID NO: 6, anHCDR3 of SEQ ID NO: 8, an LCDR1 of SEQ ID NO: 14, an LCDR2 of SEQ ID NO:14, and an LCDR3 of SEQ ID NO: 16, at a concentration of about 50mg/ml±7.5 mg/mL; (ii) histidine at 10 mM±1.5 mM, which buffers at pH6.0±0.3; (iii) sucrose at 10% w/v±1.5% w/v; and (iv) polysorbate 20 at0.2% w/v±0.03% w/v.

According to one embodiment, the pharmaceutical formulation comprises:(i) a human IgG1 antibody that specifically binds to human Ang-2, andwhich comprises a heavy chain variable domain of SEQ ID NO: 18, and alight chain variable domain of SEQ ID NO: 20, at a concentration of 25mg/ml±3.75 mg/mL; (ii) histidine at 10 mM±1.5 mM, which buffers at pH6.0±0.3; (iii) sucrose at 10% w/v±1.5% w/v; and (iv) polysorbate 20 at0.2% w/v±0.03% w/v.

According to one embodiment, the pharmaceutical formulation comprises:(i) a human IgG1 antibody that specifically binds to human Ang-2, andwhich comprises a heavy chain variable domain of SEQ ID NO: 18, and alight chain variable domain of SEQ ID NO: 20, at a concentration ofabout 50 mg/ml±7.5 mg/mL; (ii) histidine at 10 mM±1.5 mM, which buffersat pH 6.0±0.3; (iii) sucrose at 10% w/v±1.5% w/v; and (iv) polysorbate20 at 0.2% w/v±0.03%.

Additional non-limiting examples of pharmaceutical formulationsencompassed by the present invention are set forth elsewhere herein,including the working Examples presented below.

Stability and Viscosity of the Pharmaceutical Formulations

The pharmaceutical formulations of the present invention typicallyexhibit high levels of stability. The term “stable”, as used herein inreference to the pharmaceutical formulations, means that the antibodieswithin the pharmaceutical formulations retain an acceptable degree ofchemical structure or biological function after storage under definedconditions. A formulation may be stable even though the antibodycontained therein does not maintain 100% of its chemical structure orbiological function after storage for a defined amount of time. Undercertain circumstances, maintenance of about 90%, about 95%, about 96%,about 97%, about 98% or about 99% of an antibody's structure or functionafter storage for a defined amount of time may be regarded as “stable”.

Stability can be measured, inter alia, by determining the percentage ofnative antibody that remains in the formulation after storage for adefined amount of time at a defined temperature. The percentage ofnative antibody can be determined by, inter alia, size exclusionchromatography (e.g., size exclusion high performance liquidchromatography [SE-HPLC]), such that native means non-aggregated andnon-degraded. An “acceptable degree of stability”, as that phrase isused herein, means that at least 90% of the native form of the antibodycan be detected in the formulation after storage for a defined amount oftime at a given temperature. In certain embodiments, at least about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the native formof the antibody can be detected in the formulation after storage for adefined amount of time at a defined temperature. The defined amount oftime after which stability is measured can be at least 14 days, at least28 days, at least 1 month, at least 2 months, at least 3 months, atleast 4 months, at least 5 months, at least 6 months, at least 7 months,at least 8 months, at least 9 months, at least 10 months, at least 11months, at least 12 months, at least 18 months, at least 24 months, ormore. The defined temperature at which the pharmaceutical formulationmay be stored when assessing stability can be any temperature from about−80° C. to about 45° C., e.g., storage at about −80° C., about −30° C.,about −20° C., about 0° C., about 4°-8° C., about 5° C., about 25° C.,about 35° C., about 37° C., or about 45° C. For example, apharmaceutical formulation may be deemed stable if after nine months ofstorage at 5° C., greater than about 96%, 96.5%, 97%, 97.5%, 98%, 98.5%,99% or 99.5% A of native antibody is detected by SE-HPLC. Apharmaceutical formulation may also be deemed stable if after six monthsof storage at 25° C., greater than about 96%, 96.5%, 97%, 97.5%, 98%,98.5%, 99% or 99.5% of native antibody is detected by SE-HPLC. Apharmaceutical formulation may also be deemed stable if after 28 days ofstorage at 37° C., greater than about 96%, 96.5%, 97%, 97.5%, 98%,98.5%, 99% or 99.5% of native antibody is detected by SE-HPLC. Apharmaceutical formulation may also be deemed stable if after 28 days ofstorage at 45° C., greater than about 93%, 94%, 95%, 96%, 97%, 98% or99% A of native antibody is detected by SE-HPLC. A pharmaceuticalformulation may also be deemed stable if after six months of storage at−20° C., greater than about 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or99.5% of native antibody is detected by SE-HPLC. A pharmaceuticalformulation may also be deemed stable if after six months of storage at−30° C., greater than about greater than about 96%, 96.5%, 97%, 97.5%,98%, 98.5%, 99% or 99.5% of native antibody is detected by SE-HPLC. Apharmaceutical formulation may also be deemed stable if after six monthsof storage at −80° C., greater than about 97%, 97.5%, 98%, 98.5%, 99% or99.5% of native antibody is detected by SE-HPLC.

Stability can be measured, inter alia, by determining the percentage ofantibody that forms in an aggregate within the formulation after storagefor a defined amount of time at a defined temperature, wherein stabilityis inversely proportional to the percent aggregate that is formed. Thepercentage of aggregated antibody can be determined by, inter alia, sizeexclusion chromatography (e.g., size exclusion high performance liquidchromatography [SE-HPLC]). An “acceptable degree of stability”, as thatphrase is used herein, means that at most 6% of the antibody is in anaggregated form detected in the formulation after storage for a definedamount of time at a given temperature. In certain embodiments anacceptable degree of stability means that at most about 6%, 5%, 4%, 3%,2%, 1%, 0.5%, or 0.1% of the antibody can be detected in an aggregate inthe formulation after storage for a defined amount of time at a giventemperature. The defined amount of time after which stability ismeasured can be at least 2 weeks, at least 28 days, at least 1 month, atleast 2 months, at least 3 months, at least 4 months, at least 5 months,at least 6 months, at least 7 months, at least 8 months, at least 9months, at least 10 months, at least 11 months, at least 12 months, atleast 18 months, at least 24 months, or more. The temperature at whichthe pharmaceutical formulation may be stored when assessing stabilitycan be any temperature from about −80° C. to about 45° C., e.g., storageat about −80° C., about −30° C., about −20° C., about 0° C., about 4°-8°C., about 5° C., about 25° C., about 35° C., about 37° C. or about 45°C. For example, a pharmaceutical formulation may be deemed stable ifafter nine months of storage at 5° C., less than about 2%, 1.75%, 1.5%,1.25%, 1%, 0.75%, 0.5%, 0.25%, or 0.1% of the antibody is detected in anaggregated form. A pharmaceutical formulation may also be deemed stableif after six months of storage at 25° C., less than about 2%, 1.75%,1.5%, 1.25%, 1%, 0.75%, 0.5%, 0.25%, or 0.1% of the antibody is detectedin an aggregated form. A pharmaceutical formulation may also be deemedstable if after 28 days of storage at 45° C., less than about 4%, 3.5%,3%, 2.5%, 2%, 1.5%, 1%, 0.5%, or 0.1% of the antibody is detected in anaggregated form. A pharmaceutical formulation may also be deemed stableif after three months of storage at −20° C., −30° C., or −80° C. lessthan about 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1%, 0.5%, or 0.1% of theantibody is detected in an aggregated form.

Stability can be measured, inter alia, by determining the percentage ofantibody that migrates in a more acidic fraction during ion exchange(“acidic form”) than in the main fraction of antibody (“main chargeform”), wherein stability is inversely proportional to the fraction ofantibody in the acidic form. While not wishing to be bound by theory,deamidation of the antibody may cause the antibody to become morenegatively charged and thus more acidic relative to the non-deamidatedantibody (see, e.g., Robinson, N., Protein Deamidation, PNAS, Apr. 16,2002, 99(8):5283-5288). The percentage of “acidified” antibody can bedetermined by ion exchange chromatography (e.g., cation exchange highperformance liquid chromatography [CEX-HPLC]). An “acceptable degree ofstability”, as that phrase is used herein, means that at most 52% of theantibody is in a more acidic form detected in the formulation afterstorage for a defined amount of time at a defined temperature. Incertain embodiments an acceptable degree of stability means that at mostabout 52%, 50%, 45%, 40%, 35%, 30%, 29%, 28%, 27%, 26%, 25%, 20%, 15%,10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody can be detectedin an acidic form in the formulation after storage for a defined amountof time at a given temperature. The defined amount of time after whichstability is measured can be at least 2 weeks, at least 28 days, atleast 1 month, at least 2 months, at least 3 months, at least 4 months,at least 5 months, at least 6 months, at least 7 months, at least 8months, at least 9 months, at least 10 months, at least 11 months, atleast 12 months, at least 18 months, at least 24 months, or more. Thetemperature at which the pharmaceutical formulation may be stored whenassessing stability can be any temperature from about −80° C. to about45° C., e.g., storage at about −80° C., about −30° C., about −20° C.,about 0° C., about 4°-8° C., about 5° C., about 25° C., or about 45° C.For example, a pharmaceutical formulation may be deemed stable if afterthree months of storage at −80° C., −30° C., or −20° C. less than about29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%,15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or0.1% of the antibody is in a more acidic form. A pharmaceuticalformulation may also be deemed stable if after nine months of storage at5° C., less than about 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%,18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, 0.5% or 0.1% of the antibody is in a more acidic form. Apharmaceutical formulation may also be deemed stable if after 28 days ofstorage at 25° C., less than about 30%, 29%, 28%, 27%, 26%, 25%, 24%,23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody is in a moreacidic form. A pharmaceutical formulation may also be deemed stable ifafter 28 days of storage at 37° C., less than about 37%, 36%, 35%, 34%,33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%,19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, 1%, 0.5% or 0.1% of the antibody is in a more acidic form. Apharmaceutical formulation may also be deemed stable if after 28 days ofstorage at 45° C., less than about 52%, 51%, 50%, 49%, 48%, 47%, 46%,45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%,31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%,17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,0.5% or 0.1% of the antibody can be detected in a more acidic form.

Measuring the binding affinity of the antibody to its target may also beused to assess stability. For example, a formulation of the presentinvention may be regarded as stable if, after storage at e.g., −80° C.,−30° C., −20° C., 5° C., 25° C., 37° C., 45° C., etc. for a definedamount of time (e.g., 14 days to 6 months), the anti-Ang-2 antibodycontained within the formulation binds to Ang-2 with an affinity that isat least 84%, 90%, 95%, or more of the binding affinity of the antibodyprior to said storage. Binding affinity may be determined by any method,such as e.g., ELISA or plasmon resonance. Biological activity may bedetermined by an Ang-2 activity assay, such as by contacting a cell thatexpresses Ang-2 with the formulation comprising the anti Ang-2 antibody.The binding of the antibody to such a cell may be measured directly,such as via FACS analysis. Alternatively, the downstream activity of theAng-2 system may be measured in the presence of the antibody, andcompared to the activity of the Ang-2 system in the absence of antibody.In some embodiments, the Ang-2 may be endogenous to the cell. In otherembodiments, the Ang-2 may be ectopically expressed (i.e., heterologousexpression) in the cell.

Additional methods for assessing the stability of an antibody informulation are demonstrated in the Examples presented below.

Containers and Methods of Administration

The pharmaceutical formulations of the present invention may becontained within any container suitable for storage or administration ofmedicines and other therapeutic compositions. For example, thepharmaceutical formulations may be contained within a sealed andsterilized plastic or glass container having a defined volume such as avial, ampule, syringe, cartridge, bottle, or IV bag. Different types ofvials can be used to contain the formulations of the present inventionincluding, e.g., clear and opaque (e.g., amber) glass or plastic vials.Likewise, any type of syringe can be used to contain or administer thepharmaceutical formulations of the present invention.

The pharmaceutical formulations of the present invention may becontained within “normal tungsten” syringes or “low tungsten” syringes.As will be appreciated by persons of ordinary skill in the art, theprocess of making glass syringes generally involves the use of a hottungsten rod which functions to pierce the glass thereby creating a holefrom which liquids can be drawn and expelled from the syringe. Thisprocess results in the deposition of trace amounts of tungsten on theinterior surface of the syringe. Subsequent washing and other processingsteps can be used to reduce the amount of tungsten in the syringe. Asused herein, the term “normal tungsten” means that the syringe containsgreater than or equal to 500 parts per billion (ppb) of tungsten. Theterm “low tungsten” means that the syringe contains less than 500 ppb oftungsten. For example, a low tungsten syringe, according to the presentinvention, can contain less than about 490, 480, 470, 460, 450, 440,430, 420, 410, 390, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50,40, 30, 20, 10 or fewer ppb of tungsten.

The rubber plungers used in syringes, and the rubber stoppers used toclose the openings of vials, may be coated to prevent contamination ofthe medicinal contents of the syringe or vial, or to preserve theirstability. Thus, pharmaceutical formulations of the present invention,according to certain embodiments, may be contained within a syringe thatcomprises a coated plunger, or within a vial that is sealed with acoated rubber stopper. For example, the plunger or stopper may be coatedwith a fluorocarbon film. Examples of coated stoppers or plungerssuitable for use with vials and syringes containing the pharmaceuticalformulations of the present invention are mentioned in, e.g., U.S. Pat.Nos. 4,997,423; 5,908,686; 6,286,699; 6,645,635; and 7,226,554, thecontents of which are incorporated by reference herein in theirentireties. Particular exemplary coated rubber stoppers and plungersthat can be used in the context of the present invention arecommercially available under the tradename “FluoroTec®”, available fromWest Pharmaceutical Services, Inc. (Lionville, Pa.). FluoroTec® is anexample of a fluorocarbon coating used to minimize or prevent drugproduct from adhering to the rubber surfaces.

According to certain embodiments of the present invention, thepharmaceutical formulations may be contained within a low tungstensyringe that comprises a fluorocarbon-coated plunger.

The pharmaceutical formulations can be administered to a patient byparenteral routes such as injection (e.g., subcutaneous, intravenous,intramuscular, intraperitoneal, etc.) or percutaneous, mucosal, nasal,pulmonary or oral administration. Numerous reusable pen or autoinjectordelivery devices can be used to subcutaneously deliver thepharmaceutical formulations of the present invention. Examples include,but are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™,OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis,Frankfurt, Germany). Examples of disposable pen or autoinjector deliverydevices having applications in subcutaneous delivery of a pharmaceuticalcomposition of the present invention include, but are not limited to theSOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and theKWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks,Calif.), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey,L.P.), and the HUMIRA™ Pen (Abbott Labs, Abbott Park, Ill.).

The use of a microinfusor to deliver the pharmaceutical formulations ofthe present invention is also contemplated herein. As used herein, theterm “microinfusor” means a subcutaneous delivery device designed toslowly administer large volumes (e.g., up to about 2.5 mL or more) of atherapeutic formulation over a prolonged period of time (e.g., about 10,15, 20, 25, 30 or more minutes). See, e.g., U.S. Pat. No. 6,629,949;U.S. Pat. No. 6,659,982; and Meehan et al., J. Controlled Release46:107-116 (1996). Microinfusors are particularly useful for thedelivery of large doses of therapeutic proteins contained within highconcentration (e.g., about 100, 125, 150, 175, 200 or more mg/mL) orviscous solutions.

In one embodiment, the pharmaceutical formulation is administered via anIV drip, such that the formulation is diluted in an IV bag containing aphysiologically acceptable solution. In one embodiment, pharmaceuticalcomposition is a compounded sterile preparation in an intravenousinfusion bag, such that a single dose of drug product is diluted into100 mL, 250 mL (or other like amount suitable for intravenous dripdelivery) of a physiological buffer (e.g., 0.9% saline). In someembodiments, the infusion bag is made of a polyvinyl chloride (e.g.,VIAFLEX, Baxter, Deerfield, Ill.). In some embodiments, the infusion bagis made of a polyolefin (EXCEL IV Bags, Braun Medical Inc., Bethlehem,Pa.).

Therapeutic Uses of the Pharmaceutical Formulations

The pharmaceutical formulations of the present invention are useful,inter alia, for the treatment, prevention or amelioration of any diseaseor disorder associated with Ang-2 activity, including diseases ordisorders mediated by Ang-2. Exemplary, non-limiting diseases anddisorders that can be treated or prevented by the administration of thepharmaceutical formulations of the present invention include variousdiseases involving angiogenesis, which is the biological process wherebynew blood vessels are formed. Aberrant angiogenesis is associated withseveral disease conditions including, e.g., proliferative retinopathies,rheumatoid arthritis and psoriasis. In addition, it is well establishedthat angiogenesis is critical for tumor growth and maintenance.

EXAMPLES

The following examples are presented so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by mole, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric pressure.

Initial formulation development activities involved empiricalexperiments and screening organic cosolvents, thermal stabilizers, andbuffers in liquid and lyophilized formulations of anti-Ang-2 antibodiesto identify excipients that are compatible with the protein and enhanceits stability, while maintaining near physiologic osmolality and lowviscosity for intravenous and subcutaneous injection. Buffer conditionswere also examined to determine the optimal pH for maximum proteinstability.

Example 1 Development of Anti-Ang-2 Formulation

Formulation development activities included the screening of buffers,organic cosolvents, and thermal stabilizers in liquid formulations ofthe anti-Ang-2 antibody to identify excipients that enhance thestability of the protein. Buffer conditions were also examined todetermine the optimal pH for maximum protein stability. Resultsgenerated from these studies were used to develop a stable, liquidformulation suitable for clinical use. Anti-Ang-2 (e.g., HIH685P) wasformulated at 25±3.75 mg/ml and 50±7.5 mg/ml. In one embodiment, theanti-Ang-2 antibody is formulated in 10±1.5 mM histidine (pH 6.0±0.3),0.2%±0.03% polysorbate 20, 10%±1.5% sucrose.

Example 2 Buffer and pH

The effect of pH and buffer type on the stability of anti-Ang-2 wasconsidered. HIH685P at 25 mg/mL was incubated at 45° C. in eitheracetate (pH 5.0-5.5), citrate (pH 5.5-6.0), succinate (pH 6.0),histidine (pH 5.5-6.5), phosphate (pH 6.0-8.0), or Tris (pH 8.0) bufferto assess the effect of buffer and pH on the thermal stability of theprotein (Table 1). Analysis of protein stability by size exclusionchromatography (SE-HPLC) revealed that acetate (pH 5.5), phosphate (pH6.0), histidine (pH 5.5-6.5), and succinate performed better than theother buffer systems for the formulation of HIH685P. Maximum proteinstability, as determined by cationic exchange chromatography (CEX HPLC),was observed when HIH685P was formulated in histidine buffer at pH 6.0.These analyses also revealed that degradation for the antibody resultedmostly in the formation of aggregates, cleavage products, and chargevariants. The optimal pH for HIH685P stability in histidine buffer wasobserved to be 6.0, although good stability was observed in the pH rangeof 5.5 to 6.5. Based on these results, 10 mM histidine buffer at pH 6.0was chosen for the formulation of antibody drug product (DP).

TABLE 1 Effect of Buffer and pH on Stability of HIH685P Incubated at 45°C. for 28 Days OD % Total % Native % Aggregate % Main Peak % Acidic Peak% Basic Peak pH/Buffer 405¹ (RP-HPLC) (SE-HPLC) (SE-HPLC) (CEX-HPLC)(CEX-HPLC) (CEX-HPLC) Starting 0.00 100 96.4 2.5 59.6 28.9 11.5Material² pH 8.0, Tris 0.25 95 73.0 20.7 5.8 89.2 5.1 pH 8.0, 0.54 9552.7 40.2 2.4 94.0 3.6 Phosphate pH 7.5, 0.33 97 66.5 27.8 5.3 94.7 0.0Phosphate pH 7.0, 0.05 101 85.5 10.8 16.8 77.5 5.7 Phosphate pH 6.5,0.01 100 90.1 6.1 26.0 67.0 7.0 Phosphate pH 6.0, 0.00 100 92.3 4.0 31.057.5 11.5 Phosphate pH 6.5, 0.01 100 92.6 4.3 31.5 58.7 9.8 Histidine pH6.0, 0.01 100 92.3 4.3 34.9 52.2 13.0 Histidine pH 5.5, 0.00 100 92.44.0 33.1 50.4 16.5 Histidine pH 6.0, 0.00 99 92.3 4.5 32.3 56.4 11.4Succinate pH 6.0, 0.03 100 89.3 7.4 31.1 57.6 11.3 Citrate pH 5.5, 0.05100 88.6 8.0 27.3 58.7 14.1 Citrate pH 5.5, 0.00 101 92.8 3.5 32.5 54.313.2 Acetate pH 5.0, 0.00 100 91.6 3.7 29.5 52.1 18.4 Acetate

For the results shown in Table 1, 0.35 mL of 25 mg/mL HIH685P in 10 mMtest buffer in a 2 mL Type 1 borosilicate glass vial with a FluoroTec®coated 4432/50 butyl rubber stopper were tested for 28 days at 45.¹Turbidity was reported as the relative change in OD at 405 nm ascompared to the starting material. ²SE-HPLC and CEX-HPLC StartingMaterial Results were the average of the starting material for all 14formulations. OD=Optical density; RP-HPLC=Reverse phase high performanceliquid chromatography; SE-HPLC=Size exclusion high performance liquidchromatography; CEX-HPLC=cation exchange high performance liquidchromatography.

Example 3 Selection of Protectants Against Agitation Stress

The HIH685P anti-Ang-2 antibody (i.e., Drug Substance or “DS”) exhibitedlimited stability when subjected to agitation stress. Turbidity analysisof agitated DS demonstrated an increase in optical density (OD) at 405nm when HIH685P was vortexed for 120 minutes (Table 2, see No Cosolventdata). This increase in turbidity indicates a significant formation ofparticulates as a result of the agitation stress. Agitation of theformulation in the absence of cosolvent also resulted in a significantincrease in aggregate formation. Formulation with any of the evaluatedcosolvents prevented the agitation dependent increase in turbidity andaggregate levels of HIH685P (Table 2). However, the addition of 0.2%Pluronic F68, 20% PEG 300, 10% PEG 300, and 20% propylene glycol to theformulation significantly decreased the thermal stability of HIH685P asdetermined by SE-HPLC and CEX-HPLC (Table 3). Formulations containingpolysorbate 20, polysorbate 80, and PEG 3350 had no significant effecton the thermal stability of HIH685P as determined by SE-HPLC andCEX-HPLC, making these cosolvents suitable for the formulation ofHIH685P (Table 3). Polysorbate 20 was chosen as the organic cosolventfor development of a HIH685P formulation because it stabilized theprotein to agitation stress, had no effect on its thermal stability, andrequired lower co-solvent concentrations to stabilize the proteincompared to polyethylene glycol.

For the antibody stability results shown in Table 2, 0.8 mL of 5 mg/mLHIH685P, in 10 mM histidine, pH 6.0 to 6.1 in a 2 mL Type 1 borosilicateglass vial with a FluoroTec® coated 4432/50 butyl rubber stopper wascombined with the organic cosolvents and subjected to 120 minutes ofvortexing. ¹Turbidity was reported as the relative change in OD at 405nm as compared to the starting material. ²SE-HPLC and CEX-HPLC StartingMaterial Results were the average of the starting material for all nineformulations. OD=Optical density; RP-HPLC=Reverse phase high performanceliquid chromatography; SE-HPLC=Size exclusion high performance liquidchromatography.

TABLE 2 Effect of Organic Cosolvents on Antibody Stability - AgitationOrganic OD % Total % Native % Aggregate % Main Peak % Acidic Peak %Basic Peak Cosolvent 405¹ (RP-HPLC) (SE-HPLC) (SE-HPLC) (CEX-HPLC)(CEX-HPLC) (CEX-HPLC) Starting Material² 0.00 100 98.3 0.6 59.3 32.8 7.9(no vortexing) No Cosolvent 0.25 103 76.6 22.3 60.4 30.9 8.7 0.2%Polysorbate 20 0.00 99 98.3 0.6 59.5 32.9 7.7 0.2% Polysorbate 80 0.0099 98.2 0.6 59.4 32.8 7.8 0.2% Pluronic F68 0.00 98 98.3 0.7 59.4 32.77.9 3.0% PEG 3350 0.00 96 98.6 0.6 59.4 32.9 7.7 1.0% PEG 3350 0.00 9998.3 0.7 59.7 32.8 7.6 20% PEG 300 0.01 101 98.1 0.8 57.6 33.1 9.3 10%PEG 300 0.01 100 97.9 0.9 59.0 32.7 8.2 20% Propylene Glycol 0.00 10198.1 0.8 59.7 32.5 7.8

TABLE 3 Effect of Organic Cosolvents on Antibody Stability - Thermal OD% Total % Native % Aggreg. % Main Peak % Acidic Peak % Basic PeakOrganic Cosolvent 405¹ (RP) (SE) (SE) (CEX) (CEX) (CEX) StartingMaterial² 0.00 100 98.3 0.6 59.3 32.8 7.9 (no incubation) No Cosolvent0.01 97 95.3 0.7 35.1 53.8 7.3 0.2% Polysorbate 20 0.01 97 95.3 0.7 34.953.7 7.4 0.2% Polysorbate 80 0.01 97 95.1 0.9 34.9 53.8 7.5 0.2%Pluronic F68 0.01 96 95.1 0.6 31.2 53.1 9.3 3.0% PEG 3350 0.01 96 95.70.8 35.0 52.4 7.9 1.0% PEG 3350 0.01 96 96.0 0.7 35.5 52.7 7.6 20% PEG300 0.12 103 71.5 5.0 5.1 81.5 0.0 10% PEG 300 0.06 100 91.6 1.5 5.676.6 0.0 20% Propylene Glycol 0.00 98 92.2 0.8 34.3 52.5 8.2

For the antibody stability results shown in Table 3, 0.35 mL of 5 mg/mLHIH685P, in 10 mM histidine, pH 6.0 to 6.1 in a 2 mL Type 1 borosilicateglass vial with a FluoroTec® coated 4432/50 butyl rubber stopper wascombined with the organic cosolvents and subjected to 45° C. for 28days. ¹Turbidity was reported as the relative change in OD at 405 nm ascompared to the starting material. ²SE-HPLC and CEX-HPLC StartingMaterial Results were the average of the starting material for all nineformulations. OD=Optical density; RP=Reverse phase high performanceliquid chromatography; SE=Size exclusion high performance liquidchromatography; CEX=cation exchange high performance liquidchromatography.

Example 4 Selection of Protectants Against Thermal Stress

Stabilizers such as sugars, amino acids, and inorganic salts wereexamined for their ability to increase the thermal stability of HIH685P.A summary of the thermal stabilizers that were examined is presented inTable 4. Formulations containing 20% sucrose, 10% mannitol, and 20%trehalose had the least amount of HIH685P degradation as determined bySE-HPLC and CEX-HPLC analysis following thermal stress. However,formulation with mannitol destabilized the protein to multiple freezingand thawing cycles. HIH685P had similar stability to thermal stress whenformulated with sucrose or trehalose. Sucrose was chosen as the thermalstabilizer for development of a liquid HIH685P formulation.

HIH685P exhibited maximal stability when formulated in the presence ofhistidine, polysorbate 20, and sucrose at pH 6.0.10% sucrose was chosenfor the HIH685P DP formulation, which is close to iso-osmolar. The maindegradation pathways identified during the development of the HIH685Pliquid formulation were the formation of aggregates, cleavage products,and charge variants, with the highest rate of degradation (increase incharge variants) being observed when the protein was incubated at 45° C.

For the antibody stability results shown in Table 4, 0.35 mL of 10 mMHistidine, pH 6.0 to 6.2, 0.2% Polysorbate 20, and 25 mg/mL HIH685P,plus the indicated thermal stabilizer, in a 2 mL Type 1 borosilicateglass vial with a FluoroTec® coated 4432/50 butyl rubber stopper weresubjected to 45° C. for 28 days. ¹Turbidity was reported as the relativechange in OD at 405 nm as compared to the starting material. ²SE-HPLCand CEX-HPLC Starting Material Results represents the average values ofthe starting material for all ten formulations not incubated at 45° C.OD=Optical density; RP=Reverse phase high performance liquidchromatography; SE=Size exclusion high performance liquidchromatography; CEX=cation exchange high performance liquidchromatography.

TABLE 4 Effect of Thermal Stabilizers on the Stability Thermal OD 405 %Total % Native % Aggregate % Main Peak % Acidic Peak % Basic PeakStabilizer nm¹ (RP-HPLC) (SE-HPLC) (SE-HPLC) (CEX-HPLC) (CEX-HPLC)(CEX-HPLC) Starting 0.00 100 97.7 1.5 58.7 31.5 9.8 Material² NoStabilizer 0.02 100 92.9 3.7 32.0 55.2 12.8 150 mM NaCl 0.11 101 79.617.5 35.4 50.1 14.4 10% Sucrose 0.01 100 93.9 2.7 32.7 54.3 13.0 20%Sucrose 0.01 101 94.2 2.4 33.5 53.4 13.2 20% Sorbitol 0.10 101 94.4 1.924.5 62.7 12.9 10% Mannitol 0.01 99 94.4 2.3 33.1 53.1 13.8 20%Trehalose 0.01 100 94.7 2.0 33.6 52.9 13.6 5% Glycerol 0.09 103 88.0 8.14.7 92.1 3.1 3% Arginine 0.05 99 83.0 13.5 35.2 46.4 18.5 3% Glycine0.01 102 93.7 3.0 29.1 58.2 12.7

Example 5 Stable Liquid Pharmaceutical Formulation

In conclusion, HIH685P DP was produced as a liquid in an optimized,aqueous buffered formulation containing 10 mM histidine, pH 6.0, 0.2%(w/v) polysorbate 20, 10% (w/v) sucrose, and 25 mg/mL HIH685P. HIH685PDP at 25 mg/mL was physically and chemically stable when subjected tovarious stress conditions (Table 5). There was no effect on pH,appearance, turbidity, or the amount of HIH685P recovered when the DPwas vortexed, frozen and thawed multiple times, or incubated at 25° C.for 14 days. After 28 days of incubation at 37° C., the DP was 0.6% moredegraded relative to a control, unstressed sample, as determined bySE-HPLC analysis and 10.1% more degraded (decrease in % main peak) asdetermined by CEX-HPLC analysis. After 28 days of incubation at 45° C.,the DP was 3.6% more degraded compared to unstressed control asdetermined by SE-HPLC analysis and 23.8% more degraded (decrease in %main peak) as determined by CEX-HPLC analysis. No significant loss ofpotency, as determined using the HIH685P binding assay described below,was observed for any of the stressed samples.

TABLE 5 Stress Stability of 25 mg/mL Anti-Ang-2 Antibody Stress Test No45° C. 37° C. 25° C. Freeze/ Stress Agitation Incubation IncubationIncubation Thaw Length of Stress 0 60 120 14 28 14 28 14 28 8 min minmin days days days days days days cycles OD 405 nm¹ 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % Total 100 100 99 101 103 101 103 101 10297 (RP-HPLC) % Native 97.0 97.1 96.9 95.6 93.4 96.6 96.4 96.8 97.1 96.8(SE-HPLC) % Aggregate 1.9 1.9 1.9 1.8 3.5 1.6 1.7 1.6 1.7 1.6 (SE-HPLC)% Main Peak 56.0 56.1 55.9 42.8 32.2 50.4 45.9 55.1 53.5 55.7 (CEX-HPLC)% Acidic Peak 27.0 26.9 27.0 39.2 51.5 30.8 37.0 27.3 29.1 27.2(CEX-HPLC) % Basic Peak 17.0 17.0 17.1 18.0 16.3 18.8 17.1 17.7 17.417.1 (CEX-HPLC) Binding Assay² 105 NP 111 NP 99 NP 122 NP 107 110 (%Relative Potency)

Potency of the formulated HIH685P was determined by measuring theability of HIH685P to block the binding of a humanangiopoietin-2-His-tagged fusion protein (hAng2-His) to a plate coatedwith a human Tie2-murine Fc-fusion protein (hTie2-mFc) via a competitionsandwich ELISA (the binding assay). hAng2-His was titrated with varyingamounts of HIH685P. hAng2-His concentration was calculated assuming amonomeric molecular weight of 50.1 kDa. The ligand-antibody complexeswere incubated for 1 hour at 25° C. before transfer to microtiter platescoated with the hTie2-mFc. After one-hour incubation, the wells werewashed and bound hAng2-His was detected with an HRP-conjugated anti-Histag monoclonal antibody. The calculated IC₅₀ value, defined as theconcentration of antibody required to block 50% of His-tagged hAng2 toTie2-mFc, was used as an indicator of blocking potency.

For the antibody stability results shown in Table 5, 0.35 mL of 10 mMHistidine, pH 6.0 to 6.1, 0.2% Polysorbate 20, 10% Sucrose, and 25 mg/mLHIH685P, in a 2 mL Type 1 borosilicate glass vial with a FluoroTec®coated 4432/50 butyl rubber stopper was combined with the organiccosolvents and subjected to the various designated stresses.⁴⁰⁵¹Turbidity was reported as the relative change in OD at 405 nm ascompared to the starting material. ²The acceptance criteria for thebinding assay was 50-150% of reference standard. OD=Optical density;RP=Reverse phase high performance liquid chromatography; SE=Sizeexclusion high performance liquid chromatography; CEX=cation exchangehigh performance liquid chromatography.

Example 6 Stability of Formulated Anti-Ang-2 Antibody

Stability studies were performed to determine both the storage andstress stability of the anti-Ang-2 antibody formulation containing 25mg/mL antibody, 10 mM histidine, pH 6.0±0.3, 0.2% polysorbate 20, and10% sucrose. Turbidity and RP-HPLC assays were used to assess thephysical stability of the antibody. Physical stability is defined as therecovery of soluble forms of the anti-Ang-2 antibody in solution. Lossof protein could be due to either protein precipitation or surfaceadsorption. The presence of particulates in solution can be detected byvisual inspection or by optical density (OD) measurements at 405 nm(turbidity measurements). In this latter assay, an increase in ODindicates an increase in turbidity due to the formation of particulates.The presence of particulates as determined by OD measurements indicatesthat the sample has failed to maintain stability. Recovery of antibodyis measured by RP-HPLC. In the RP-HPLC assay, the anti-Ang-2 antibody iseluted from the reverse phase column as a single peak. The concentrationof each test sample is determined from the area of the eluted antibodypeak compared to a calibration curve generated using antibody standardsof defined protein loads.

Chemical stability refers to the integrity of the chemical structure ofthe anti-Ang-2 antibody in a sample. Most chemical instability can beattributed to the formation of covalently modified forms of theantibody, (e.g. covalent aggregates, cleavage products, or chargevariants) and non-covalently modified forms of the antibody (e.g.non-covalent aggregates). Thus far, the only degradation products ofHIH685P that have been detected are species that differ in eithermolecular weight or charge. The higher and lower molecular weightdegradation products can be separated from native antibody by SE-HPLC.The percentage of native in the size exclusion chromatographic method isdetermined by the ratio of the area of the native peak to the total areaof all anti-Ang-2 antibody peaks.

Charge variant forms of the anti-Ang-2 antibody were resolved fromnative antibody using cation exchange chromatography. Peaks that elutedfrom the CEX-HPLC column with retention times earlier than that of themain peak were labeled “Acidic Peaks”, while those that eluted from theCEX-HPLC column with retention times later than that of the main peakwere labeled “Basic Peaks”. The percentage of degraded anti-Ang-2antibody in the cation exchange chromatographic method is determined bythe change in the relative percentage of the main, acidic, and basicpeak areas compared to the total area of all anti-Ang-2 antibody peaks.

Evaluation of the antibody under accelerated conditions was performed bysubjecting the antibody to a variety of stress tests. These testsrepresent the extreme handling conditions that the formulated drugsubstance may be subjected to during the manufacture, storage, ortransportation of drug product. The formulated anti-Ang-2 antibody wasfilled in 5 mL polycarbonate vials for the agitation, cycles offreeze/thaw, and frozen storage conditions. The formulated antibody wasfilled in glass vials to examine stress stability at high temperatures.

Example 7 Storage Stability

No change was observed in the physical and chemical stability of the 25mg/mL HIH685P antibody formulation containing 10 mM histidine, pH6.0±0.3, 0.2% polysorbate 20, and 10% sucrose, when stored for sixmonths at −80° C., −30° C., −20° C., and 5° C., and for nine months at5° C. A slight diminution in binding activity (ELISA competition assaydescribed above) was observed at 6 months at −80° C., −30° C., and −20°C., i.e., ˜86%, ˜84%, and ˜91% of baseline, respectively. See Table 6.

Example 8 Stress Stability

The stress stability of the 25 mg/mL anti-Ang-2 antibody (HIH685P)formulated in 10 mM histidine, pH 6.0±0.3, 0.2% polysorbate 20, 10%sucrose was discerned by subjecting the formulation to agitation,thermal stress (45° C., 37° C., 25° C.), and freeze thaw stress. The0.35 mL of the formulation was filled in a 2 mL Type 1 borosilicateglass vial fitted with a FLUROTEC-coated 4432/50 butyl rubber stopper.The results are presented in Table 7.

Example 9 Molecular Mass Determination

A series of analytical, biochemical and biophysical techniques were usedto characterize the formulated HIH685P antibody. The molecular weightsof the heavy and light chains of glycosylated and deglycosylated HIH685Pantibody samples were determined following CapillaryElectrophoresis-Sodium Dodecyl Sulfate (CE-SDS) analysis under reducingconditions. Samples were denatured in 0.5% w/v sodium dodecyl sulfate(SDS) and 40 mM dithiothreitol (DTT) at 60° C. for 10 min. Followingdenaturation and reduction, 1×G7 buffer v/v (1% Nonidet 40 and 50 mMsodium phosphate pH 7.5) was added to the samples. Deglycosylation ofHIH685P (50 μg) was accomplished by addition of 1250 units of PeptideN-glycosidase F (PNGase F) and incubation for three hours at 37° C.Untreated control samples were prepared similarly except that the PNGaseF enzyme was omitted from the three hour incubation. Samples weredesalted following incubation to remove components of the reactionbuffer that may interfere with CE-SDS analysis. Desalted samples werecompletely denatured in 1% w/v SDS and 4.5% w/v β-mercaptoethanol andincubated at 80° C. for 10 min. A 10 kDa molecular weight standard(Beckman Coulter) was added to each sample and used as an internalstandard to determine peak identity and calculate protein mobility.

TABLE 6 Stability of 25 mg/mL HIH685P antibody in 10 mM Histidine, pH6.0, 0.2% Polysorbate 20, 10% Sucrose Storage −80° C./ −30° C./ −20° C./5° C./ 5° C./ Condition 6 mo. 6 mo. 6 mo. 6 mo. 9 mo. Appearance PassPass Pass Pass Pass pH 6.1 6.1 6.1 6.0 6.1 Turbidity 0.00 0.00 0.00 0.000.00 (OD 405 nm)¹ % Total 105 105 107 102 105 REGN910 Recovered(RP-HPLC) Purity by Size- Exclusion- HPLC % main peak 97.3 96.8 96.897.1 96.6 purity % HMW species 1.8 1.9 1.9 1.8 1.7 Charged VariantAnalysis by CE-HPLC % region 1 28.3 28.1 27.9 28.1 27.4 (acidic) %region 2 (main) 55.5 55.9 55.8 55.6 56.3 % region 3 16.2 16.0 16.3 16.416.2 (basic) Binding Assay 85.95 84.3 90.9 >100 NP (% Ref. Std.)

Three principal peaks were observed in the electrophoregrams of theHIH685P untreated control samples. Peak 1 represents reduced light chainwith a calculated molecular weight of approximately 28 kDa (34.0-34.1%total peak area). Peaks 2 and 3 represent non-glycosylated heavy chain(˜51 kDa; 3.1-3.8%) and glycosylated heavy chain (˜56 kDa; 62.2-62.8%total peak area), respectively. The sum of heavy and light chains (peaks1, 2, and 3) represent ≧99% of the total percentage of peaks detected bythis method for both HIH685P lot samples.

TABLE 7 Stress Stability Stress Test No 45° C. 37° C. 25° C. Freeze/Stress Agitation Incubation Incubation Incubation Thaw Length of Stress60 120 14 28 14 28 14 28 8 — min min days days days days days dayscycles Appearance Pass Pass Pass Pass Pass Pass Pass Pass Pass Pass pH6.1 6.0 6.1 6.1 6.2 6.1 6.1 6.0 6.1 6.1 Turbidity 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 (OD at 405 nm) % Total REGN910 100 100 99101 103 101 103 101 102 97 Recovered (RP-HPLC) Purity by SE- HPLC 97.097.1 96.9 95.6 93.4 96.6 96.4 96.8 97.1 96.8 % main peak purity % HMWspecies 1.9 1.9 1.9 1.8 3.5 1.6 1.7 1.6 1.7 1.6 Charged Variant 27.026.9 27.0 39.2 51.5 30.8 37.0 27.3 29.1 27.2 Analysis by CEX-HPLC %region 1 (acidic) % region 2 (main) 56.0 56.1 55.9 42.8 32.2 50.4 45.955.1 53.5 55.7 % region 3 (basic) 17.0 17.0 17.1 18.0 16.3 18.8 17.117.7 17.4 17.1 Binding Assay 100 NP >100 NP 94.3 NP >100 NP >100 >100 (%Relative Potency)

In the samples treated with PNGase F, there was a substantial decreasein the intensity of peak 3 coupled with a concomitant increase in theintensity of peak 2 in each electropherogram, indicating removal of theglycan chain from the glycosylated heavy chain. The average correctedpeak area percentage of peak 2 from the electropherograms generatedfollowing capillary electrophoresis of PNGase F treated HIH685P lotsamples (66%) was equivalent to the sum of the mean peak areapercentages of peaks 2 and 3 from the electropherograms generatedfollowing capillary electrophoresis of the control, non-PNGase F treatedsamples (66%), indicating complete deglycosylation of the heavy chainfollowing PNGase F incubation. As expected, the molecular weight andrelative migration time of the light chain was unchanged.

Multi-angle laser light scattering (MALLS) is an analytical method thatprovides an estimate of the molecular mass of a protein or glycoprotein.The molar mass of native HIH685P was analyzed by connecting the flowcell of a light scattering detector to a gel filtration column to allowmolecular mass analysis of each separated drug substance component(SEC-MALLS). The protein concentration was monitored by differentialrefractive index and absorbance detectors. Formulated HIH685P lotsamples were injected directly onto a TSK Gel G3000SWxI (TosohBiosciences, cat 08541; column dimensions 0.78 cm×30 cm, 5 μm particlesize, and porosity of 250 Å), pre-equilibrated in 10 mM sodiumphosphate, 500 mM NaCl, pH 6.1 buffer (SEC buffer). The data wereanalyzed by Astra software (Wyatt Technology) using the specificrefractive index increment, which is defined as the change in refractiveindex divided by the change in protein concentration (dn/dc), correctedfor the high ionic strength of the mobile phase buffer. By SEC-MALLSanalysis, the main peak (peak 4, elution volume of ˜7.9 mL) for HIH685Pcorresponded to a molecular weight of approximately 151 kDa. The molarmass identified for the main peak (peak 4) corresponded to intactanti-Ang-2 antibody, which was the predominant species present insolution (96.1-96.9% of the total protein peak area). A very smallpercentage of two high molecular weight species, corresponding to peak 2(elution volume of ˜6.2 mL) and peak 3 (elution volume of 6.5 mL) andtwo low molecular weight species, corresponding to peak 5 (elutionvolume of ˜8.4 mL) and peak 6 (elution volume of ˜9.8 mL), were detectedin all formulated HIH685P samples tested. The calculated molar mass frompeak 3 (elution volume of ˜6.5 mL) of approximately 300 kDa isconsistent with a dimeric form of the HIH685P antibody, and represents asmall fraction (1.5-1.9%) of the total antibody molecular weight formspresent in solution. The calculated molecular weight for peak 5 (elutionvolume of ˜8.4 mL) is approximately 66 kDa and represents ≦1.5% of thetotal molecular weight forms present in the sample. Peak 6 represents˜0.2-0.3% of the total peak area. The results from SEC-MALLS analysis ofthe HIH685P drug substance demonstrated that the majority of the proteinexists as an intact antibody with an average molar mass of approximately151 kDa.

Independent lots of formulated HIH685P were further analyzed to confirmthe molecular weight of the intact proteins through mass measurement.1.5 μg of each protein sample was injected onto a 1.7 μm BEH130 C18column coupled to a Waters Synapt Mass Spectrometer for massmeasurement. The ESI-TOF mass spectra were deconvoluted for the intactproteins using a maximum entropy algorithm with 11 iterations. Based onthe heavy and light chain cDNA sequences of HIH685P, the intact antibody(with heavy chain C-terminal Lys removed) was predicted to have amolecular weight of 144604.4 Da. The deconvoluted mass spectra of theintact HIH685P lots showed a similar pattern, with each spectracontaining multiple peaks differing by masses of either 146 dalton(fucose) or 162 dalton (galactose), suggesting the presence ofglycosylation related micro-heterogeneity. These glycan masses wereobtained by subtracting the predicted molecular weight of 144604.4 fromthe observed intact masses. For example, the 2nd ESI/MS peak has m/z147493 Da, less of intact MAb MW at 144604.4 Da leaves the glycan massat 2889 Da. Since the glycans are added on the both sides of Fc, theglycan mass should be 1445 Da.

These glycan masses were used to assign the major HIH685P glycanstructural forms. The analysis showed that these glycans are primarilycomposed of fucosylated complex bi-antennary structures, with 0, 1, and2 galactoses at the glycan chain termini. The mass difference betweenthe predicted and observed masses (4 Dalton) for the protein amino acidsequence was within the mass accuracy specification of the Synapt MS.These results confirmed the identity of HIH685P at the primary sequencelevel.

Example 10 Isoelectric Point Determination

HIH685P was analyzed by one-dimensional isoelectric focusing (IEF) undernative conditions to determine the isoelectric point (pI) of the intactantibody. A series of pI standards were included in the study, withprotein bands visualized by Colloidal blue staining. All samplepreparations of HIH685P exhibited a total of eight bands of varyingintensity that migrated between a pI of 7.9 and 8.4, with the dominantform having a pI of 8.2. The band migrating at a pI of approximately8.2, exhibited a slightly lower pI than the predicted pI of 8.6 for theintact antibody. This major band most likely represents fullyglycosylated intact antibody lacking the C-terminal lysine (predicted pIof 8.54). This interpretation is consistent with mass spectrometryanalysis of the heavy chain following proteolytic digestion and reversephase separation. In the mass spectrometry analysis, the dominantC-terminal peptide from the HIH685P heavy chain was observed to belacking the terminal lysine residue, predicted to be present from thecDNA sequence. Three minor basic variants (corresponding to a pI rangeof 8.3-8.4) were observed. While not wishing to be bound by theory,these minor species may represent partial forms of the antibody, such asan antibody form possessing only heavy chain dimer (predicted pI of8.9), or heavy chain dimer lacking one or two C-terminal lysines.Alternatively, these species may be consistent with a small amount of acharged variant form of the intact antibody containing one or twoC-terminal lysines. This interpretation is consistent with the massspectral analysis of the heavy chain after tryptic digestion andseparation of peptides by reverse phase chromatography. Approximately2.5% of the HIH685P heavy chain was identified by mass spectrometry tocontain the C-terminal lysine.

The pI of the native, monomeric form of the kappa light chain fromHIH685P is predicted to be 6.4. No band corresponding to a pI of 6.4 wasdetected under the conditions employed, suggesting that minimal, if any,amounts of free light chain are present in the formulated HIH685Ppreparation. Although currently unidentified, four additional minorbands that migrated to a more acidic pH relative to the principal band(corresponding to a pI range of 7.9-8.1) were present in all testedsamples, and may be speculated to represent intact, glycosylatedantibody with deamidated forms, incorrectly formed intermoleculardisulfide bonds, or small amounts of truncated forms of the antibody.

The charge heterogeneity of formulated HIH685P was also quantitativelyassessed by isoelectric focusing a capillary electrophoresis (cIEF)based method. For this study, each HIH685P sample was diluted to 0.4mg/mL (100 μg) in cIEF gel (Beckman Coulter, cat 477497) containing 39mM arginine, 2.3 mM iminodiacetic acid, and 3-10 Pharmalytes. (GEHealthcare; 12 μL). A total of seven peaks of varying intensity wereobserved ranging from 7.77 to 8.48. The pI range of product relatedpeaks observed by cIEF is slightly greater than the pI range observed bythe gel-based isoelectric focusing method (pI range of 7.9-8.4) due tothe increased resolution of the capillary. Highly similar qualitativepeak patterns were observed from the electropherograms from each DS lottested. There were a total of four major peaks (peaks 4-7) thatrepresented approximately 92-93% of the total peak area. While notwishing to be bound by theory, these peaks may represent intact antibodylacking the C-terminal lysine, truncated or partial forms of theantibody, non-covalent intact forms of the antibody, or forms withincorrectly formed disulfides. It is possible that some of these peaksmay arise during sample preparation or under the electrophoreticconditions employed. Peak 4 represents the dominant charge variantspecies with a pI of approximately 8.1 and average peak area percentagesof 57.5% and 53.4% for each of two formulated HIH685P sample lotstested.

To further examine charge heterogeneity of the HIH685P formulatedantibody, several samples were analyzed by two-dimensional gelelectrophoresis. For the 2-D gel method, proteins were reduced and thenseparated on the basis of charge in the first dimension usingisoelectric focusing. Subsequently, separated charged species werefurther resolved in the second dimension based on molecular weight usingSDS-PAGE performed under reducing conditions. Each reduced HIH685Psample (2 μg), in the presence or absence of Bio-Rad 2-D pI standards,was loaded onto pH 3-10 immobilized pH gradient (IPG) strips foranalysis in the first dimension. Following isoelectric focusing, the IPGstrips were loaded onto 4-20% Novex Tris glycine gels for analysis inthe second dimension. The second dimension was run in the presence ofmolecular weight standards. In addition, a series of internal standardsof known pI were included in the isoelectric focusing analysis. Allproteins were visualized by Coomassie Blue staining. Following 2-D gelelectrophoresis and protein spot analysis, all HIH685P lot samplesexhibited comparable spot patterns, with similar charge variant speciespresent, likely due to equivalent heterogeneity of the heavy chain. Atotal of three HIH685P-related spots were observed in the gel thatcorresponded to an approximate mass of 55 kDa, with one highly abundantform exhibiting a pI corresponding to approximately 8.2. This spot (pIof ˜8.2) is consistent with the reduced, glycosylated heavy chainpolypeptide lacking the C-terminal lysine, detected by mass spectrometryanalysis. Two unidentified minor spots, detected in the 2-D gel,exhibited isoelectric points between approximately pH 7.5-8.0. In onetheoretical scenario, it is possible that these spots consist of assaydependent deamidated protein forms or truncated forms of the heavychain. Alternatively, it is possible that sample processing or gelartifacts may have led to spot spreading, resulting in the appearance ofadditional minor spots of varying isoelectric points.

As anticipated, the HIH685P light chain migrated within the 2 D gel as asingle major spot, corresponding to a pI of approximately 6.0 with amolecular weight of approximately 25 kDa. An unidentified minor spot (pIof ˜5.8) with a molecular weight of about 25 kDa was also observed. Thisspot may represent a small percentage of deamidated light chain, whichwas also detected in mass spectrometry analysis.

Example 11 Post-Translational Modifications

Like most IgG1 antibodies, the HIH685P heavy chain terminates with theexpected amino acid sequence, Pro-Gly-Lys, with the C-terminal Lysresidue susceptible to removal by an unidentified basic carboxypeptidaseduring protein expression. Partial removal of the terminal Lys couldlead to charge heterogeneity within the antibody drug substance. Fromthe peptide mapping analysis of the reduced HIH685P, it was confirmedthat the majority of the C-terminal Lys⁴⁵² residue in the trypticpeptide was removed from the heavy chain, resulting in a C-terminalpeptide with a sequence of ⁴⁴⁵SLSLSPG⁴⁵¹ (SEQ ID NO: 532). Only a verysmall percentage of tryptic peptide containing the C-terminal Lysresidue (⁴⁴⁵SLSLSPGK⁴⁵²; SEQ ID NO: 533) was observed in the trypticmap. Based on the integrated peak areas of the two tryptic C-terminalheavy chain peptides from each lot of HIH685P, approximately 1.0% and1.5% of the HIH685P antibody heavy chain contained C-terminal Lys⁴⁵² inthe toxicology and clinical lots, respectively.

Non-enzymatic deamidation of asparagine is another common modificationthat is frequently observed in antibodies. Deamidation, which occurs viathe formation of a succinimide intermediate, followed by hydrolysis,results in the formation of isoaspartate and aspartate. Each deamidationevent introduces one additional negative charge to an antibody andgenerates charge heterogeneity. An asparagine residue followed byglycine or serine within the primary amino acid sequence is by far themost susceptible site for deamidation within a protein. In HIH685P, twoasparagine residues in the heavy chain (Asn³²⁰ and Asn³⁸⁹) are directlyfollowed by glycine residues and are therefore candidate sites fordeamidation. Mass spectrometry analysis indicated that littledeamidation occurred at Asn³²⁰ in any of the HIH685P sample lots.Focused mass analysis revealed that Asn³⁸⁹ was identified within apeptide eluted as peak 53. A peptide containing a deamidated form ofAsn³⁸⁹ eluted within peak 52. Due to other co-eluting peptides withinpeak 52, it was not possible to obtain a quantitative analysis of therelative amount of deamidation based on the UV signal. However, usingion intensities from the mass spectra, the relative percentage ofdeamidated Asn³⁸⁹ in either of the lots of HIH685P was determined to beless than 2%. In the reduced peptide map of HIH685P, mass spectrometryanalysis also identified peak 49 as a light chain tryptic peptide thatcontained a native form of asparagine 94 followed by serine. Anequivalent peptide, containing a deamidated form of Asn⁹⁴ from the lightchain, was also identified (peak 48). Integration of these two peaksrevealed that approximately 4.2% of the total amount of this asparaginewas present in the deamidated form in both the toxicology and clinicallots. Light chain asparagine 94 was identified as the only Asn-Sercontaining peptide that exhibited deamidation end products in HIH685P.The light chain Asn⁹⁴ is located within the thirdcomplementarity-determining region (CDR). Due to the structurallydisruptive conditions used to prepare the peptide mapping samples, itcannot be ruled out that the deamidation observed at Asn⁹⁴ may have beengenerated during the tryptic digestion procedure. No deamidation wasobserved at any other asparagine residue to any detectable level. Theresult suggests that deamidation of asparagine is not expected to occurto any appreciable level during the manufacture of HIH685P.

The potential for oxidation of surface methionine residues, which canpotentially affect HIH685P stability and activity, was also examined.HIH685P contains three methionine residues located within the heavychain. Some of the methionine residues are predicted to be located onthe surface of the antibody making them susceptible for oxidation.Oxidation of the methionine side chain to the sulfide form increases theside chain mass by 16 daltons, and makes the side chain more polar.Extensive mass data analysis, focused on the methionine-containingpeptides, revealed no oxidation at any of the three methionine residuesin any of the tested lots of HIH685P, suggesting that expression andpurification processes as well as storage conditions of the drugsubstances does not result in chemical changes to the protein.

Example 12 Glycosylation Patterns

Glycosylation is a major post-translational modification that can leadto molecular mass heterogeneity of antibodies. Human IgG1 isotypeantibodies contain a single canonical asparagine-linked (N-linked)glycosylation site located within the heavy chain constant region (Fcdomain). Analysis of the reduced HIH685P tryptic map identified twoglycopeptides corresponding to two tryptic peptides from the Fc domain(amino acid sequences of ²⁹⁸EEQFNSTYR³⁰⁶ (SEQ ID NO: 534) and²⁹⁴TKPREEQFNSTYR³⁰⁶ (SEQ ID NO: 535)). The major glycan forms on theseglycopeptides were elucidated based on MS analysis, and are summarizedin Table 8. Glycopeptide mass analysis revealed that the HIH685PN-linked sugar is predominantly composed of a complex, bi-antennarystructure, with a core fucose and zero, one, or two galactose residuesat the glycan chain termini. These structures are consistent with thetypical glycan forms found on recombinant antibodies expressed frommammalian cells. The LC/MS analysis also revealed a peptide containingthe Fc glycosylation site (Asn³⁰²) but lacking glycan occupancy (peak 18and peak 23). Integration of the peak areas in the UV chromatogramsshowed the 4.8% and 5.2% of N-linked sites at the Fc exhibited no glycanoccupancy in the toxicology and clinical lots, respectively. Theseresults are in agreement with the capillary electrophoresis analysis.The MS analysis of each of the peptides generated from the tryptic mapreveals that HIH685P has no other N-linked or O linked glycosylationsites within the HIH685P antibody molecule.

TABLE 8 Peptide Assignments From Tryptic Map Observed Expected RetentionPeptide Peptide Peak Time Fragment Mass^(d) Mass^(e) No.^(a) (min)^(b)Identity^(c) (Da) (Da) Comments 1 8.8 H220-223 471.294 471.269 2 9.0H340-343 447.287 447.269 3 9.9 H326-331 734.390 734.374 4 11.3 H219-223599.390 599.364 5 11.9 L184-188 624.303 624.275 6 12.9 L184-190 889.461889.429 7 15.1 L208-211 522.279 522.255 8 15.3 H361-365 604.334 604.3069 17.2 H216-223 941.611 941.554 10 18.0 L19-24 706.372 706.343 11 18.5L208-214 868.376 868.349 12 19.2 L104-107 487.331 487.300 13 20.3H415-421 817.490 817.465 14 20.4 H415-419 574.348 574.332 15 20.6L104-108 643.428 643.401 16 21.0 H298-306 2957.177 2957.144Fuc(GlcNAc)₂(Man)₃(GlcNAc)₂(Gal)₂ H298-306 2795.150 2795.091Fuc(GlcNAc)₂(Man)₃(GlcNAc)₂(Gal) H298-306 2592.198 2592.011Fuc(GlcNAc)₂(Man)₃(GlcNAc)₁(Gal)₁ H298-306 2429.972 2429.959Fuc(GlcNAc)₂(Man)₃(GlcNAc) H298-306 2405.031 2404.927 (GlcNAc)₂(Man)₅ 1721.1 H298-306 2633.086 2633.038 Fuc(GlcNAc)₂(Man)₃(GlcNAc)₂ H298-3062487.066 2486.980 (GlcNAc)₂(Man)₃(GlcNAc)₂ H298-306 2283.996 2283.901(GlcNAc)₂(Man)₃(GlcNAc) H298-306 2267.995 2267.917Fuc(GlcNAc)₂(Man)₂(GlcNAc) H298-306 2226.959 2226.879 Fuc(GlcNAc)₂(Man)₃H298-306 2121.904 2121.848 (GlcNAc)₂(Man)₂(GlcNAc) H298-306 2080.9572080.821 (GlcNAc)₂(Man)₃ 18 22.0 H298-306 1188.547 1188.505non-glycosylated 19 22.6 L56-62 728.413 728.381 20 23.0 H294-3063277.467 3277.387 Fuc(GlcNAc)₂(Man)₃(GlcNAc)₂(Gal)₁ H294-306 3439.4923439.440 Fuc(GlcNAc)₂(Man)₃(GlcNAc)₂(Gal)₂ 21 23.1 H294-306 3115.4113115.335 Fuc(GlcNAc)₂(Man)₃(GlcNAc)₂ H294-306 2969.325 2969.277(GlcNAc)₂(Man)₃(GlcNAc)₂ H294-306 2766.276 2766.198(GlcNAc)₂(Man)₃(GlcNAc)₁ 22 23.8 L146-149 559.340 559.311 23 24.2H294-306 1670.876 1670.801 non-glycosylated 24 27.9 L150-169 2135.0242134.961 25 28.8 H67-75 1064.579 1064.561 26 29.5 H87-97 1245.5891245.544 27 29.9 H65-75 1277.733 1277.683 28 30.6 H65-71 835.491 835.46629 30.9 H445-452 787.493 787.443 Heavy Chain with C-terminal K (1.5%) 3032.8 H445-451 659.364 659.349 Heavy Chain C-terminal K Removal (=98.5%)31 33.2 H254-260 834.459 834.426 H332-339 837.515 837.495 32 35.9L189-207 2140.139 2140.073 33 37.0 L191-207 1874.980 1874.919 34 37.4L184-207 2746.385 2746.338 35 38.0 L47-55 978.584 978.549 36 40.6H344-365 2509.374 2509.328 L146-169 2676.313 2676.262 37 41.1 H350-3651872.004 1871.962 H139-152 1320.696 1320.670 38 44.6 H366-375 1160.6801160.622 H127-138 1185.692 1185.639 39 45.8 L170-183 1501.801 1501.75040 46.3 L63-78 1631.823 1631.778 L25-46 2452.250 2452.213 H1-19 1881.0521880.995 41 47.2 H76-86 1337.715 1337.675 42 47.9 L150-183 3618.7803618.701 H280-293 1676.822 1676.794 43 48.5 L1-18 1883.018 1882.999 4448.9 H261-279 2138.056 2138.019 45 49.8 H420-444 3043.452 3043.392 4650.1 H422-444 2800.328 2800.259 47 51.0 L56-78 2342.233 2342.150 48 51.9L79-103 2997.316 2997.300 N₉₄S partial deamidation (4.2%) 49 52.3L79-103 2996.373 2996.292 50 52.7 L146-183 4160.162 4161.003 51 53.0L79-108 3621.812 3621.683 52 53.4 H376-397 2544.165 2544.131 N₃₈₉Gpartial deamidation 53 53.7 H376-397 2543.220 2543.123 54 54.6 H39-642651.450 2651.322 55 55.0 H350-375 3014.715 3014.574 56 56.3 H398-4141872.973 1872.914 H44-66 2166.157 2166.063 57 57.7 H20-38 2203.0602203.015 58 58.5 L109-126 1945.065 1945.019 59 61.5 H224-253 3333.6963333.634 60 62.8 H228-253 2843.510 2843.450 61 63.6 H307-325 2227.2542227.199 62 64.2 L127-142 1796.938 1796.887 63 65.3 H307-322 1807.0421806.999 64 65.7 H228-260 3659.800 3659.866 65 66.4 H376-414 4398.0824398.027 66 69.5 L150-183 3618.780 3618.701 67 73.1 H153-223 7635.9627635.851 68 73.5 H153-219 7182.596 7182.592 69 74.0 H153-218 7054.6037054.497 70 74.4 H153-215 6712.410 6712.307 71 76.4 H139-223 8938.5818938.511 72 76.8 H139-219 8485.280 8485.252 73 77.3 H139-218 8357.1368357.157 74 77.6 H139-215 8014.698 8014.967 75 78.9 H98-126 1985.6202985.548 ^(a)Column 1: Peak numbers corresponding to the chromatographicpeak numbers. ^(b)Column 2: Retention time of each peptide peak inminutes. ^(c)Column 3: Location of peptide within the HIH685P sequences.H and L denote sequences located within HIH685P heavy chain and lightchain, respectively. ^(d)Column 4: Experimentally determined peptidemass from LC/MS and LC/MS/MS analysis. ^(e)Column 5: Theoretical peptidemass calculated from predicted trypsin digestion cleavage sites withinthe amino acid sequence of HIH685P sequence.

What is claimed is:
 1. A pharmaceutical formulation comprising: (i) anantibody that binds specifically to human angiopoietin 2 (human Ang-2);(ii) a buffer at pH of 6.0±0.3; (iii) a non-ionic detergent; and (iv) astabilizer.
 2. The pharmaceutical formulation of claim 1, wherein theantibody comprises an HCDR1 of SEQ ID NO: 4, an HCDR2 of SEQ ID NO: 6,an HCDR3 of SEQ ID NO: 8, an LCDR1 of SEQ ID NO: 12, an LCDR2 of SEQ IDNO: 14, and an LCDR3 of SEQ ID NO:
 16. 3. The pharmaceutical formulationof any one of claims 1 and 2, wherein the antibody comprises a HCVD ofSEQ ID NO: 18 and an LCVD of SEQ ID NO:
 20. 4. The pharmaceuticalformulation of claim 2, wherein (a) over 96% of the antibodies have amolecular weight of 151 kDa±1 kDa; (b) at least 53% of the antibodieshave an isoelectric point of about 8.13±0.01; and (c) from about 90% toabout 92% of the antibodies are fucosylated.
 5. The pharmaceuticalformulation of claim 4, wherein the antibody concentration is about 25mg/mL±0.375 mg/mL, or about 50 mg/mL±7.5 mg/mL.
 6. The pharmaceuticalformulation of claim 5, wherein the buffer is histidine.
 7. Thepharmaceutical formulation of claim 6, wherein the histidineconcentration is 10 mM±1.5 mM.
 8. The pharmaceutical formulation ofclaim 7, wherein the non-ionic detergent is polysorbate
 20. 9. Thepharmaceutical formulation of claim 8, wherein the polysorbate 20concentration is about 0.2% w/v±0.03%.
 10. The pharmaceuticalformulation of claim 9, wherein the stabilizer is sucrose.
 11. Thepharmaceutical formulation of claim 10, wherein the sucroseconcentration is about 10%±1.5% (w/v).
 12. The pharmaceuticalformulation of claim 2, wherein the antibody concentration is 25mg/mL±3.75 mg/mL, the buffer is 10 mM±1.5 mM histidine, pH 6±0.3, thenon-ionic detergent is 0.2% w/v±0.03%, and the stabilizer is 10%w/v±1.5% sucrose.
 13. The pharmaceutical formulation of claim 2, whereinthe antibody concentration is 50 mg/mL±7.5 mg/mL, the buffer is 10mM±1.5 mM histidine, pH 6±0.3, the non-ionic detergent is 0.2%w/v±0.03%, and the stabilizer is 10% w/v±1.5% sucrose.
 14. Thepharmaceutical formulation of claim 2, wherein at least 93% of theantibody has native conformation after 28 days at 45° C.
 15. Thepharmaceutical formulation of claim 2, wherein at least 32% of theantibody is the main charge variant of the antibody after 28 days at 45°C.
 16. The pharmaceutical formulation of claim 2, wherein at least 97%of the antibody has native conformation after 28 days at 25° C.
 17. Thepharmaceutical formulation of claim 2, wherein at least 53% of theantibody is the main charge variant of the antibody after 28 days at 25°C.
 18. The pharmaceutical formulation of claim 2, wherein at least 96%of the antibody has native conformation after 28 days at 37° C.
 19. Thepharmaceutical formulation of claim 2, wherein at least 45% of theantibody is the main charge variant of the antibody after 28 days at 37°C.
 20. The pharmaceutical formulation of claim 2, wherein at least 97%of the antibody has native conformation after six months at 5° C. 21.The pharmaceutical formulation of claim 2, wherein at least 55% of theantibody is the main charge variant of the antibody after six months at5° C.
 22. The pharmaceutical formulation of claim 2, wherein the percentrelative potency of the antibody after six months at 5° C. is at least100% of the potency of the antibody prior to storage.
 23. Thepharmaceutical formulation of claim 2, wherein at least 97% of theantibody has native conformation after six months at −80° C.
 24. Thepharmaceutical formulation of claim 2, wherein at least 55% of theantibody is the main charge variant of the antibody after six months at−80° C.
 25. The pharmaceutical formulation of claim 2, wherein thepercent relative potency of the antibody after six months at −80° C. isat least 85% of the potency of the antibody prior to storage.
 26. Thepharmaceutical formulation of claim 2, wherein at least 96% of theantibody has native conformation after six months at −30° C.
 27. Thepharmaceutical formulation of claim 2, wherein at least 55% of theantibody is the main charge variant of the antibody after six months at−30° C.
 28. The pharmaceutical formulation of claim 2, wherein thepercent relative potency of the antibody after six months at −30° C. isat least 84% of the potency of the antibody prior to storage.
 29. Thepharmaceutical formulation of claim 2, wherein at least 96% of theantibody has native conformation after six months at −20° C.
 30. Thepharmaceutical formulation of claim 2, wherein at least 55% of theantibody is the main charge variant of the antibody after six months at−20° C.
 31. The pharmaceutical formulation of claim 2, wherein thepercent relative potency of the antibody after six months at −20° C. isat least 90% of the potency of the antibody prior to storage.
 32. Apharmaceutical formulation comprising (a) 25 mg/mL mL±3.75 mg/mL of ananti-Ang-2 antibody, (b) 10 mM±1.5 mM histidine, pH 6±0.3, (c) 0.2%w/v±0.03% polysorbate 20, and (d) 10% w/v±1.5% sucrose, wherein: (a) theantibody comprises an HCVD of SEQ ID NO: 18 and an LCVD of SEQ ID NO:20; (b) over 96% of the antibodies have a molecular weight of 151 kDa±1kDa; (c) at least 53% of the antibodies have an isoelectric point ofabout 8.13±0.01; and (d) from about 90% to about 92% of the antibodiesare fucosylated.
 33. The pharmaceutical formulation of claim 32consisting of (a) 25 mg/mL±3.75 mg/mL of the antibody, (b) 10 mM±3 mMhistidine, pH 6±0.3, (c) 0.2% w/v±0.03% polysorbate 20, and (d) 10%w/v±1.5% sucrose, in water.
 34. A pharmaceutical formulation comprising(a) 50 mg/mL mL±7.5 mg/mL of an anti-Ang-2 antibody, (b) 10 mM±1.5 mMhistidine, pH 6±0.3, (c) 0.2% w/v±0.03% polysorbate 20, and (d) 10%w/v±1.5% sucrose, wherein: (a) the antibody comprises an HCVD of SEQ IDNO: 18 and an LCVD of SEQ ID NO: 20; (b) over 96% of the antibodies havea molecular weight of 151 kDa±1 kDa; (c) at least 53% of the antibodieshave an isoelectric point of about 8.13±0.01; and (d) from about 90% toabout 92% of the antibodies are fucosylated.
 35. The pharmaceuticalformulation of claim 34 consisting of (a) 50 mg/mL mL±7.5 mg/mL of theantibody, (b) 10 mM±1.5 mM histidine, pH 6±0.3, (c) 0.2% w/v±0.03%polysorbate 20, and (d) 10% w/v±1.5% sucrose, in water.
 36. Apharmaceutical composition of claim 11, wherein said composition iscontained in a container.
 37. The pharmaceutical composition of claim36, wherein the container is a vial.
 38. The pharmaceutical compositionof claim 37, wherein the vial is glass.
 39. The pharmaceuticalcomposition of claim 36, wherein the container is an IV drip bag. 40.The pharmaceutical composition of claim 39, wherein the bag is made ofpolyvinyl chloride.
 41. The pharmaceutical composition of claim 39,wherein the bag is made of polyolefin.
 42. A kit comprising apharmaceutical composition of any one of claim 11, a container, andinstructions.
 43. The kit of claim 42, wherein the container is a glassvial fitted with a FLUROTEC-coated 4023/50 rubber stopper.